The common mucosal immune system for the reproductive tract: basic principles applied toward an AIDS vaccine

advanced

drug delivery reviews

Advanced

Drug

D&very

Reviews

IX ( 1995) 23-Sl

The common mucosal immune system for the reproductive tract: basic principles applied toward an AIDS vaccine Hiroshi Kiyono”‘*, Christopher J. Mille?, Yichen Lu’, Thomas Lehnerd Martin Cranaged, Yung T. Huange, Shigetada Kawabata”, Marta Marthask, Bryan RobertsC, John G. Nedrud”, Michael E. Lamm”, Lesley Bergmeierd, Roger Brookesd, Louisa Tao”, Jerry R. McGhee”

“Collahortrtive

Mtccosul

Immtmization

Research

Group,

Califbrnia

Primate

Research

Cerzter. Utziver\ity

of C‘ali,fbmin.

Da\,is.

C‘A Y.5616 USA ‘Collaborative

Mucosal

Immuni;ation

“Collaborative United ‘Collaborative

Medical Mtrcosal

and Dental

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Mucosal Schools

Immunization

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(rt GLIY‘S Hospital.

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Research Centw

Institute

OH

44106.

Accepted

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of

Applied

of Pathology,

Irxtitute. Division

Cambridge,

MA

02/.~Y. USA

of immunology,

Microbiology, Case Western

London Reserve

SEI

YRl:

Urtiversit~v.

UK Cleveland.

USA

June

1995

Abstract The concept of the Collaborative Mucosal Immunization Research Group for AIDS (CMIG) was originally conceived by the AIDS Vaccine Branch, National Institute of Allergy and Infectious Diseases (NIAID) in order to provide support for a cooperative research environment for the development of mucosal immunity to AIDS. We have purposely organized five groups of investigators at five different locations to determine how effective mucosal immunity to AIDS can be optimally approached. CMIG recognizes that both rectal (homosexual) as well as vaginal (heterosexual) infections with HIV are two of the major ways that AIDS currently disseminates through the human population. Thus, we have chosen the SIVmodel of infection of rhesus macaques, but more importantly the CMIG have joined two of our five components in order to use the significant expertise developed for mucosal transmission of SIV and immunity. Thus, we have brought the extensive expertise with vaginal and rectal immunization and immunity to spread [Drs. Chris Miller and Marta Marthas, California Regional Primate Research Center (CRPRC), Davis and Drs. Thomas Lehner and Martin Cranage, United Medical and Dental School Guy’s Hospital. London and the Centre for Applied Microbiology and Research (Guy’s/CAMR)]. Two additional components were added in order to perform mucosal immune response studies required to develop and to optimize a mucosal vaccine. First. extensive CD4’ T helper (Th) cell (e.g., Thl and Th2) and CDS’ T cell subset studies are a major effort of the coordinating group at the University of Alabama at Birmingham (Drs. Hiroshi Kiyono and Jerry R. McGhee). This component is closely interacting with both the CRPRC and Guy’s/CAMR components in terms of SIV-specific CD4. and CD8’ T cell subset responses. For example, SIV-specific CTL responses are jointly examined using different techniques by CRPRC, Guy’s/CAMR and UAB investigators. Further, it is also important to examine a balance between SIV-specific and Thl and Th2 cell responses following mucosal immunization since the Th cell-derived cytokines are essential for the induction of appropriate antigen-specific * Cormqxmding

author

0169-409X/95/$29.00 (Q 1995 Elsevier SSDl 0169-409X(95)00049-6

Science

B.V. All rights

reserved

24

tt. Klyono

et al. i Acivuncrcl Drug Delivery

Reviews

IX (199.7) Z-51

mucosal immune responses. This issue is currently being extensively examined by the CMIG effort and a summary of our findings is discussed in this review. A major question in mucosal immunity involves the functions of secretory IgA (S-IgA) antibodies and this area is of particular importance in rectal and reproductive tract immunity. A novel and exciting in vitro epithelial cell assay system is used to study how effectively S-IgA neutralizes SIV infection (Drs. John Huang, John Nedrud and Michael Lamm, Case Western Reserve. Cleveland). A clear advantage of this CMIG effort is the unique expertise in design of mucosal delivery systems for an AIDS vaccine. We are using state-of-the-art recombinant bacteria, i.e.. rSalmonelZu and rvibrios for mucosal immunization [Drs. Yichen Lu and Bryan Roberts, Virus Research Institute (VRI), Boston]. In addition. we are also testing other mucosal delivery systems including DNA vaccine. microspheres, cholera toxin (CT) and CT-B, recombinant poliovirus, and immune complexes. These studies represent the first efforts to induce not only Th cell mediated S-IgA responses, but also CTL responses to SIV in primates immunized with different mucosal vector delivery systems. In order to focus our effort for the induction of SIV-specific immune responses following mucosal immunization, investigators from the CMIG are attempting to understand the induction and regulation of antigen-specific immune responses in rhesus macaques mucosally immunized with different preparations of SIV vaccines. Keywords: I/. cholera vector; S. typhi vector; Polyphosphazene microencapsulation: HIV-l non-replicative DNA vaccine; Mucosal immunity: Mucosal vaccine: Mucosal AIDS: Thl cell; Th2 cell

viron;

Contents 1. Introduction .................................................................................................... 2. Induction of mucosal and peripheral antibody responses ........................................................... 3. Importance of ThZ-type cells for the induction of antigen-specific mucosal immune responses ......................... 4. Mucosal immune system for the reproductive tract ................................................................. 4.1. Mucosal immunity in the female reproductive tract ........................................................... 4.1 .I. Unique immunologic characteristics of female reproductive tract tissues ............................. 4.1.2. Evidence for both mucosal and systemic immunity in the female reproductive tract ......................... 4.1.3. Inductive sites for immunity in the reproductive tract ...................................................

. .....

4.2. The distribution of lymphoid cells in the male reproductive tract ................................................ SIV infection in the reproductive tract .................................................................. 5.1. Transmission of HIV and SIV in the female reproductive tract ................................................. 5.2. Transmission of HIVand SIV in the male reproductive tract ................................................... 5.3. Hypothetical route of transmission .................................. . .......................... ............. 5.4. Importance of viral variants in sexual transmission of HIV ..................................................... 6. Anti-HIV and SIV functions of S-IgA ............................................................................ ............................................... 6.1, Evidence for HIV and SIV infection through mucosal epithelium 6.2. Can IgA block invasion of HIV and SIV at mucosal surfaces? ................................................... 6.3, Intraepithelial cell neutralization of viruses by IgA ........................................................... .............................................. 6.4. Dimeric IgA mediated excretion of virus through the epithelium 6.5. Model system for neutralization of SIV by IgA ......................................................... .. ... 7. New developments for HIV and SIV specific mucosal vaccines ...................................................... 7.1. Novel mucosal vaccine delivery vectors ..................................................................... 7.1 ,I, Live attenuated bacterial vectors ....................................................................... 7.1.2. Biodegradablemicrospheres ........................................................................... 7.1.3. Non-replicating HIV-l particles ....................................................................... 7.1.4. DNAvaccinefor HIV ................................................................................ 7.2. Induction of SWspecific immune responses in the genital. rectal and urinary tracts .................... .......... 7.2.1. Comparative quantitative analysis of p27specific IgA antibodies following five routes of immunization ...... 7 2.? Comparative proliferative responses of T lymphocytes following five routes of immunization ................ 7.2.3. Comparative CD4 ’ Th helper function in B cell antibody synthesis following five routes of immunization 7.3. Mucosal TLN immunization induced SIV-specific Thl and Th2 cells for antigen-specific IgG and IgA B cell responses .................................................................................................. Acknowledgments ................................................................................................ References ...................................... ........ ........................ ...............................

5. HIVand

25 2s 27 28 28 28 29 30 31 31 32 32 33 33 34 34 34 35 35 36 36 37 37 39 40 41 41 42 43 43 44 4s 4.5

H. Kiyono et al. I Advanced

Drug Delivery Reviews 18 (1995) 23-51

1. Introduction

The World Health Organization estimates that 6-8 million people are currently infected with HIV worldwide. The most conservative estimates predict that 15-20 million people will be infected with HIV by the year 2000 and most of these people will be infected through heterosexual contact. This is particularly true in the urban centers of sub-Saharan Africa [1,2]. Despite its importance, little is known about the biology of this mode of transmission. In developed countries, most of the acquired immune deficiency syndrome (AIDS) patients became infected with HIV through homosexual contact or the use of contaminated needles during intravenous drug abuse. However, the vast majority of presently healthy HIV-infected individuals probably became infected by heterosexual contact and are unaware of their infection status. Because the mean incubation period from seroconversion to AIDS is about 8-10 years [3], these individuals have the potential, through sexual contact, to widely disseminate HIV before they become aware that they are infectious. Thus, AIDS has the potential to become a far more serious global epidemic than is currently the case. Epidemiological studies indicate that, compared to other sexually transmitted diseases, HIV is not efficiently transmitted by sexual contact [4]. For example, 22-25% of individuals are infected by one exposure to Neisseria gonorrhoeae [5,6] and hepatitis B virus is transmitted in 20-30% of exposures [7,8]. In contrast, less than 15% of those monogamous individuals repeatedly exposed to an infected sexual partner become infected with HIV [9]. It is estimated that for a single sexual contact, the infectivity of HIV is 0.3% [lo-131. However, some individuals become infected after a single or a few sexual contacts [13,14], while others remain uninfected despite hundreds of contacts [ll]. The low transmissibility notwithstanding, it is generally agreed that the route of infection by HIV in adults is through mucus membranes. Thus, the induction of HIV-specific immunity at mucosal surfaces in addition to peripheral immunity could play an important role in resisting HIV infection. It is now established that the mucosal immune

25

system is a distinct and separate component of the host’s immune apparatus and differs from the lymphoid tissues in peripheral sites which contribute to the antibody isotypes found in the blood circulation. Furthermore, the mucosal immune system is regulated in a different fashion than that of peripheral lymphoid tissues (often referred to as the systemic immune system). The mucosal immune system can be divided into discrete inductive sites where vaccines and/or antigens are encountered and are endocytosed, processed, and presented to B and T cells (mucosal inductive sites) and to separate areas where immune cells actually secrete antibody (mucosal effector tissues) [15,16]. B-cell commitment to immunoglobulin A (IgA), the major isotype of the mucosal immune system, is thought to occur in the inductive site (e.g., Peyer’s patches; PP) prior to enteric antigen exposure. Furthermore, through the assistance provided by T helper (Th) cells and cytokines, these B cells respond to antigen and undergo expansion, including memory cell formation. Emigration of antigen-activated lymphoid cells from inductive sites to mucosal effector tissues, such as lamina propria (LP) and epithelium of the gastrointestinal (GI), upper respiratory (UR) and genitourinary tracts, precedes the B cell development of IgA plasma cells. It also appears that antigen-specific Th cells, as well as CD8’ cytotoxic T lymphocytes (CTLs) can make this circulatory journey (along with B cells) from inductive to mucosal effector sites; mucosal immunologists have termed this the common mucosal immune system (CMIS) (Fig. 1).

2. Induction of mucosal and peripheral antibody

responses

Studies from several animal models and in humans have provided rather compelling evidence that, for example, oral immunization can lead to antigen and/or vaccine uptake into the PP through specialized epithelial ceils termed M cells (reviewed in Refs. [15,16]). Interestingly, however, this antigen and/or vaccine stimulation of IgA recursor B cells [surface IgA’ (sIgA+) B cells], Th cells and CTLs does not result in full

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blown immune responses in the PP. Instead. oral immunization leads to the dissemination of antigen-specific B cells, Th cells and CTLs to mucosal effector tissues such as the LP of the CiI. UR and genitourinary tracts and to salivary, mammary and lacrimal glands for subsequent antigen-specific secretory IgA (S-IgA) responses via the CMIS [IS. 171. Further, it is important to note that oral immunization can also induce antigen-specific immune responses in systemic compartments. The results of studies showing that antigen specific B and Th cells (and possibly CTLs) populate mucosal effector sites via the CMIS have provided the rationale for development of mucosal vaccines and unique antigen delivery systems that attempt to optimize antigen uptake by PP. Perhaps the best studied system involves

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the co-administration (or direct coupling) of cholera toxin (CT) or CT-B subunit with vaccine immunogens, because CT supports good mucosal S-IgA responses and redirects mucosal and strum antibody responses to co-administered proteins [ 17-211, which often induce systemic unresponsiveness (oral tolerance) when given alone. Other major delivery systems include microspheres. which encapsulate vaccines (and thus protect them from the acidity and proteolytic environment) for subsequent uptake into IgA inductive tissues. Finally, other packaging systems include ISCOMS and liposomes, and live attenuated bacteria or viruses expressing the vaccine antigen of interest. A recently described novel approach employing plasmid DNA expressing infuenza virus antigenic proteins has been shown to provide

H. Kivono et al.

i Advanced Drug Delivery Reviews 18 (199.5) 2.3%.fl

protective immunity to challenge by the virus. Administration of the plasmids by gene-gun techniques or more conventional methods (i.e., intravenous, intramuscular, or intranasal administration) has shown promising results [22]. Following entry into the transfected cell, the DNA encoded protein is transiently expressed by the host cell, inducing an immune response. The possible beneficial aspects of such an approach versus the associated risks involved in the introduction of foreign DNA into a variety of human cells will require careful evaluation (see section 7.1 below). At present, few studies have evaluated the mucosal immune response to plasmid DNA encoded proteins exposed to mucosal surfaces. Recombinant avirulent (former pathogens) viruses (i.e., adeno- and poliovirus) and bacteria e.g., avirulent rVibrio cholerae or rSalmonella typhimurium or rBacillus Calmette-Guerin (rBCG) in mice and rS. typhi or rBCG in humans are also promising approaches to delivery of vaccine antigens in the PP for disseminated S-IgA responses in distant mucosal effector sites [16] (see details in section 7.1). In addition, commensal bacteria including oral streptococci [16a] as well as Lactobaciffus species are currently being developed as recombinant delivery systems to colonize and induce immune responses in the oral cavity and the GI tract, respectively.

3. Importance induction responses

of Th24ype cells for the of antigen-specific mucosal immune

A major tenet in mucosal immunology is that S-IgA responses to protein-based vaccines are regulated by T cells, and that CD4’ Th cells support this isotype response [15,16]. However, the majority of research which supported this assumption was derived from in vitro studies. Only recently, it has been shown that selective removal of CD4’ T cells in vivo by treatment of mice with monoclonal anti-CD4 antibodies prevents mucosal IgA responses to CT [23], and greatly diminishes the overall numbers of IgA plasma cells in mucosal effector tissues [24]. Of

27

more relevance to T cell help for IgA responses, the cytokines IL-5 and IL-6 induce post-switch S-IgA’ B cells to divide and differentiate into IgA producing cells [25-271. Thus far, few studies had attempted to correlate the induction of CD4’ Th cells in mucosal inductive sites with the profile of cytokines produced or measured antigen-induced Th cell and cytokine responses along with concomitant mucosal S-IgA, or with serum IgM, IgG or IgA antibody responses. To address this issue, the profiles of cytokines produced by CD4’ Th cells in mucosal inductive sites during optimal mucosal IgA and serum IgG and IgA responses were examined at both mRNA and single cell levels [28,29,33]. Earlier studies have shown that oral immunization with CT induced Th2-type cell responses in both PP and spleen (SP), while i.v. injection induced some Thl- and brisk Th2-type systemic responses in SP, but not PP. These findings suggested that the route of immunization is important for the induction of different subsets of Th cell responses (281. It was also shown that oral immunization with a classical T cell dependent antigen, sheep red blood cells (SRBC), preferentially induced CD4 ’ Th cells which produced IL-5 [29]. These findings further supported the hypothesis that the route of immunization and other factors such as the nature of antigen-presenting cells (APCs) present in lymphoid tissues, e.g., PP and SP, influence the Thl- and Th2-type cell response patterns. It has been shown in vitro that splenic APC stimulated the proliferation of both Thl and Th2 cell clones while hepatic non-parenchymal cells stimulated the proliferation of Thl but not Th2 cell clones [30]. Further, IFN-?, and LPS activated macrophages (M0) failed to stimulate Thl clones despite expressing high levels of MHC class II molecules and membrane IL-l; however, these M0 induced strong Th2 cell proliferative responses [31]. In contrast, Thl clones were effectively stimulated by freshly isolated peritoneal M0 activated by intraperitoneal injection of Con A or live Listeria monocytogenes [30]. Therefore, APCs most likely play an important role in determining the nature of Th cell responses. In the case of PP, this tissue has been shown to contain both functional M0 and dendritic cells

28

H. Kiyono ef al. I Advanced

Drug Delivery Reviews 1X (199-f) L-51

(DC) for antigen presentation (reviewed in Refs. [15,16]). The role of antigen-specific B cells, including memory B cells for induction of Th2 cells in mucosal inductive or effector sites, has not been studied; however, this is an area which should be thoroughly investigated. Despite accumulated evidence which suggests that CT is an effective mucosal adjuvant for the induction of IgA responses, only limited information is currently available, especially data concerning its effect on Th cell subsets for mucosal immunity. It was of major importance to determine whether CT could enhance Th2 cell responses to other protein vaccines when given by the oral route. To test this possibility, the well-characterized protein vaccine tetanus toxoid (IT) was selected as a model antigen for these studies. Mice were given various doses of IT together with 10 pg of CT and it was shown that 250 pg of TT gave maximum antibody responses [32]. In the next experiments, PP CD4’ Th cells from these mice were examined for frequencies of TT-specific Thl and Th2 cell responses. Clearly, higher numbers of TT-specific IL-4 and IL-5 spot-forming cells (SFC) were induced in PP CD4’ Th cell cultures when compared with IL-2 or IFN-y SFC [33]. These findings showed that both CT and ‘IT preferentially induce antigenspecific Th2 cell responses when given orally to mice. Taken together, these findings suggested that the induction of antigen-specific Th2 type responses in mucosa-associated tissues were essential for the generation of S-IgA immune responses. For the development of mucosal vaccines, one must consider antigen delivery systems which induce the activation of Th2 type pathways for antigen-specific S-IgA responses.

4. Mucosal immune

system of the reproductive

tract

It is well known that HIV and SIV infect cells of the immune system including CD4+ T lymphocytes, MO and DC. To understand the biology of these viruses in any local anatomic site, characterization of the distribution and function of these cell types at the site itself is required. Significant progress has been made recently in

characterizing the components of the genital immune system in males and females and this work is summarized below. 4.1. Mucosal immunity in the female reproductive tract The immune system of the female reproductive tract is currently under intensive investigation mainly because of the increased incidence of viral and bacterial sexually transmitted diseases (STDs) and the finding that >75% of new AIDS cases can result from the heterosexual spread of HIV In addition, significant efforts toward a mucosal contraceptive vaccine are well underway. In this section, we briefly review the relative contribution of the mucosal and systemic immune systems to the antibodies present in the female reproductive tract. Antibodies of various isotypes are present in cervico-vaginal secretions, and can originate from serum as well as from local synthesis by resident plasma cells. For example, antibodies have been induced by direct immunization of the vagina with protein antigens or as a result of infection of the lower reproductive tract with a variety of pathogens [34-361. The immune system in the female reproductive tract provides crucial protection against the spread of STDs; however, it is the least understood part of the mucosal immune system with respect to the origin of the resident immune cells, the role of tissue CTL responses and the induction of antibody responses as well as the contribution of serum-derived versus mucosally produced antibodies. 4.1.1. Unique immunologic characteristics of female reproductive tract tissues The mucosa of the vagina consists of nonkeratinized, stratified squamous epithelium and an underlying vascular submucosa. The ectocervix has a similar architecture whereas the endocervix consists of a simple columnar epithelium covering a vascular submucosa. The mucosal immune system in the genital tract of the female rhesus macaque consists of a resident population of monocytes/MQ) and T cells in the submucosa of the vagina and cervix [37]. These cells are specifically localized in the superficial submu-

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Drug Delivery Reviews 18 (1995) 23-51

cosa, just beneath the vaginal epithelium. A similar population of lymphocytes and M0 has been described in the submucosa of the human cervix [38]. The mechanism of antigen uptake and processing in the female reproductive tract has only recently been elucidated. M cells have not been described in the vagina or cervix but Langerhans’ cells and MO are present in the vaginal mucosa. To this end, MHC class II+, and CD4+ dendritic cells/Langerhans cells are abundant in the vaginal and ectocervical mucosa of women [38-421 and rhesus macaques [37] and dendritic processes extend to the lumen of the vagina, presumably to sample antigen. These cells are located within squamous epithelia throughout the body. They are commonly found in the skin where they can bind antigen and migrate into the draining lymph node. In the draining lymph node, Langerhans’ cells transform into interdigitating DC of the T cell-rich paracortex [43-461. In mice, CD4’ Langerhans’ cells were involved in antigen absorption in the vagina [47]. By analogy to a primary immune response in the GALT, antigen reaching the submucosa of the vagina is taken up by APCs, which then migrate to draining lymph nodes. Once in the lymph nodes, the APCs stimulate B and T lymphocytes, including memory subpopulations, that enter the bloodstream via the efferent lymph and thoracic duct. These T and B lymphocytes migrate to the genital tract and, on exposure to the antigen, participate in a secondary immune response. It is likely that Langerhans’ cells and mononuclear phagocytes present in the vagina are capable of acting as APCs for initiation of an immune response (Fig. 2). 4.1.2. Evidence for both mucosal and systemic immunity in the female reproductive tract Two types of secretions have been studies in the female genital tract: (1) secretions in the Fallopian tubes, uterus and peritoneal cavity which require invasive procedures to obtain; and (2) the more easily obtained cervical mucus (from the cervico-vaginal junction) or vaginal fluid (vaginal washes) which contain secretions from both the cervix and vagina. The presence of IgG and IgA antibodies in uterine secretions is

29

hormonally regulated and, in rats, maximal concentrations of IgG and IgA occur at prooestrus, but the concentration of IgG decreases markedly at oestrus [48]. Local inflammation also markedly influences the levels found in genital secretions. Human cervical mucus from normal women sampled throughout the menstrual cycle contained albumin: IgG ratios that approximate those in serum, clearly suggesting that the IgG is derived from the blood circulation [49]. Several groups have assessed the concentration of IgA in cervical mucus and about 85-90% is locally produced S-IgA. The effect of estrogen on local IgA synthesis in women is unclear, and one study reported a higher concentration of IgA than of IgG at mid cycle [50], whereas another showed that the concentrations of IgG and IgA were consistently lower at mid cycle [34]. These observations emphasize the importance of hormones for regulation of local antibody production in genital secretions. Indirect evidence that IgA is locally produced in the upper reproductive tract was provided by the finding of reduced IgA (but not IgG) in vaginal secretions of hysterectomized mice [51]. In hysterectomized women, the concentration of IgG in cervical mucus was 50% of normal, whereas the concentration of IgA was
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clearly suggests that the IgA produced in these regions is largely polymeric [52-541. Interestingly, approximately equal distributions of IgAI and IgA2 plasma cells were noted, and this pattern is most similar to that seen in the lower regions of the GI tract. Women with a variety of STDs have increased numbers of plasma cells (especially IgA plasma cells) in the submucosa of the endocervix [56]. Finally, epithelial cells lining the endo- and ectocervix, the vagina and the Fallopian tubes produce secretory component (SC), the polymeric Ig receptor, which is required for transport of polymeric IgAl and polymeric IgA2 into secretions of the reproductive tract. In summary, these studies clearly indicate that certain regions such as the Fallopian tubes, the ectoand endocervix and perhaps the vagina, exhibit characteristics of mucosal effector sites.

lymphatic

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4.1.3. Inductive sites for immunity in the reproductive tract Several possible inductive sites may contribute effector B and T lymphocytes to the female reproductive tract. Adoptive transfer of lymphocytes from the mesenteric lymph nodes resulted in repopulation of the cervix, vagina and uterus in the mouse [56]. The isotype distribution of Ig-containing cells was such that 60% were IgA and about 25% were IgG. The mesenteric lymph nodes are major draining sites for PP in the small intestine, and are thought to contain lymphocytes that are migrating from the PP to distant mucosal effector tissues [57]. The oestrous cycle influences B lymphoblast localization, and maximum repopulation of genital tissues by mesenteric lymph node cells occurred during pro-oestrus and oestrus [58]. No changes in the homing of B

H. Kiyono et al. I Advanced

Drug Delivery Reviews IX (199-i) 23-51

lymphoblasts into the LP of the GI tract were seen at any stage of the oestrous cycle [58]. Based on the previously mentioned studies, it is possible to speculate on the origins of B and T cells in reproductive tissues. First, the GALT may provide cells for this effector site (Fig. 1); however, it may not be a primary source for these B cells. Since the human endo- and ectocervix contain an IgAl and IgA2 distribution similar to that of the large intestine, there may be a common origin for lymphocytes populating these two sites (Fig. 1). These regional lymph nodes (obturator and iliac lymph nodes) which drain the large intestine also drain reproductive tissues. Thus, a “shunt” of the common mucosal immune system may include rectal follicles in the rectal-associated lymphoreticular tissues (RALT) of the large intestine, as additional inductive sites for the reproductive tract (Fig. 1). Since direct immunization of the vagina with antigen induces an immune response, the cells of the female lower reproductive tract can initiate an immune response. Based on the evidence to date, it seems likely that induction of immunity in the female genital tract involves antigen uptake by APCs (such as Langerhans’ cells and M0) in the reproductive tract, with antigen processing by these cells and migration of APCs to draining lymph nodes (Fig. 2). Furthermore, B cells stimulated in GALT and especially RALT may contribute significantly to the immune response and home to the reproductive tract sites (Fig. 1). To this end, it was recently shown that the targeted lymph node (TLN) route of immunization for the genitourinary-rectal associated lymphoid tissue with a vaccine consisting of SIVgag p27 resulted in the induction of antigenspecific S-IgA antibodies in genital fluids [59] (see details in section 7.2). 4.2. The distribution of lymphoid cells in the male reproductive tract A recent chapter provides an excellent description of the organization of immune cells in the male reproductive tract [60] and that discussion is briefly summarized below. There are numerous M0 but no lymphocytes in the intestitium of the testis and no white blood cells are

31

found in the seminiferous tubules of normal men. However, a variety of pathologic conditions can result in increased numbers of T cells within the testicles. In normal testicles from healthy men, CD8’ T cells are located in the columnar epithelium of the rete testis and CD4’ T cells are found in the connective tissue around the rete testis. T lymphocytes also occur in the epididymis, seminal vesicles and prostate of normal men and CD4’ T cells are found in human epididymal epithelium. In addition to T cells, numerous M0 are present in the intersititium of the excurrent ducts and accessory glands. The penile urethra also contains a complete complement of immune cells. Numerous CD4’ and CD8’ T lymphocytes, DC and M0 are present in the lamina propria and submucosa of the penile urethra (Pudney, J. and Anderson, D., personal communication). In addition, numerous Langerhans’ cells are located in the foreskin of rhesus macaques [61] and men (Miller, C., unpublished observations). Thus, like other mucosa-associated tissues, the male reproductive tract contains immunocompetent cells. In future studies, it will be important to determine whether this site is also protected by the CMIS.

5. HIV and SIV infection tract

in the reproductive

In the United States, HIV is apparently more efficiently transmitted from men to women than from women to men [12,62]. Several cofactors have been identified which increase the risk of an individual acquiring HIV through heterosexual contact. Cervical ectopy, receptive anal intercourse, genital ulcer disease and infection with other STDs are the most significant factors associated with HIV infection of women [63-651 while the presence of an intact foreskin and genital ulcer disease of the penis are the risk factors most often associated with HIV infection in men [64,65]. There are three possible explanations for the variability in the sexual transmission of HIV First, unique factors in the HIV-infected individual (stage of disease, immune response, pres-

32

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ence of other STDs) may influence infectivity. Some HIV-positive persons are highly infectious and others, presumably, are not [14,66,67]. The degree to which an individual is infectious may be related to the stage of disease. During primary HIV infection, the levels of antigen and virus in plasma are high [68-701 and this initial viremia may be associated with high levels of HIV in genital secretions. As yet studies have not been undertaken in people to test this hypothesis, but in the SIV-rhesus macaque model, SIV can be isolated from the semen of intravenously inoculated macaques as early as 10 days post-inoculation (Miller, unpublished results). HIV is more common in the semen of men with AIDS than in semen of healthy chronically infected individuals [71]. Second, unique factors in specific HIV strains may influence transmission. Some HIV strains may be inherently more likely than other strains to be shed in the secretions of infected persons or some strains may, by virtue of their cellular tropism, have an increased affinity for target cells in the reproductive tract. Third, unique factors (stage of menstrual cycle, nutritional status or the presence or absence of the risk factors listed above) may influence the susceptibility of individuals to HIV infection following exposure. Some people may be highly susceptible to HIV infection, while others may be more resistant. 5.1, Transmission of HIVand reproductive tract

SIV in the female

The identity and location of the cellular targets of HIV and SIV during genital transmission are yet to be determined. Simple application of SIV onto the intact genital mucosa of mature and immature rhesus macaques results in virus transmission and the disease induced by this route of inoculation is indistinguishable from that seen in intravenously inoculated animals [72,73]. This model is being used to understand the interaction between virus and host during the sexual transmission of HIV SIV was efficiently transmitted to hysterectomized female macaques by inoculation of cell-free virus into blind vaginal pouches [74]. The 50% infectious dose of cell-free virus was the same in the intact and hysterectomised ani-

mals, indicating that sufficient target cells are present in the intact vaginal mucosa for transmission of the virus. Since only a few CD4’ T cells are present in the submucosa of the vagina [37], the most likely target cells in the vaginal mucosa are M0 or Langerhans cells 139,751. In chronically infected female rhesus macaques, SIV-infected cells are present in the uterine cervix and vagina [75]. The majority of the SIV-infected cells are located in the submucosa of the ectocervix and vagina and have a morphology consistent with T lymphocytes and monocytes/MQ). SIV-infected cells are also found within the stratified squamous epithelium of the vagina. HIV-infected Langerhans cells have been reported in the skin of AIDS patients [76,77]. The finding that cells in the vaginal epithelium are infected with SIV is the first indication that mucosal Langerhans cells might be infected with a lentivirus [75]. In cervical biopsy material obtained from HIV-infected women, T cells and M0 were determined to be the cell types infected with HIV These findings are consistent with the results in SIV-infected monkeys. All cells that were found to be infected in the genital tract of both human and rhesus macaques presumably express the CD4 molecule. 5.2. Transmission of HIV and SIV in the male reproductive tract The transmission of HIV and SIV to males may involve similar types of target cells. Studies to detect the location of HIV-infected cells in the penis of infected men are lacking but the fact that CD4+ Langerhans cells are located in the foreskin and the epidemiological evidence indicating that the presence of an intact foreskin is associated with an increased risk of HIV infection suggests that Langerhans cells in the foreskin may be target cells during genital transmission. A recent study demonstrated that in SIV-infected rhesus macaques, these foreskin Langerhans cells contain SIV nucleic acid [61]. Adult male rhesus macaques can be infected with SIV by placing cell-free virus onto the foreskin of the animals [78]. These findings suggest that Langerhans cells may have a role as target cells in the sexual

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transmission of HIV and SIV. These APCs are potentially efficient disseminators of the virus from the mucosa of the reproductive tract to draining lymph nodes. As mentioned above, these cells function to transport antigen from the stratified squamous epithelium to the draining lymph node [43-461. Blood DC (of which Langerhans cell precursors are a subset) can be infected with HIV [79,80]. Furthermore, when infected in vitro, these cells produce much higher levels of virus than T cells but they do not exhibit the usual cytopathic effects associated with HIV infection [80]. Thus, Langerhans cells in the vagina and ectocervix [38-421 as well as foreskin [61,78] may be especially well suited as target cells for the sexual transmission of HIV and SIV [74,75].

5.3. Hypothetical route of transmission Based on these observations, we have proposed the following hypothesis to explain the cellular events in the sexual transmission of HIV and SIV [74]. The virus infects APC (M0 and Langerhans cells) in the mucosa of the reproductive tract and are carried by these cells to the iliac and obturator lymph nodes draining the genital tract. In rhesus macaques, India ink inoculated into the vaginal submucosa localizes in these lymph nodes within 72 h (Miller, unpublished results). It is likely that the virus initially replicates in the target cells (MO and Langerhans cells) and undergoes a second round of replication in the draining lymph nodes prior to spreading to more distant lymphoid tissue. In heterosexual transmission, it is likely that the initial virus-host cell interaction occurs in the genital mucosa or submucosa and the early events in the dissemination of HIV occur in the draining lymph node. Thus, to prevent dissemination of the virus to the systemic lymphoid tissue the protective immune response, both antibody and cell-mediated immunity, must be present in the genital mucosa, submucosa and in the draining lymph node. A strong systemic immune response may provide another level of protection from infection after sexual contact with an infected partner but a genital mucosal

33

immune response would concentrate the protective response at the initial sites of infection. 5.4. Importance of viral variants in sexual transmission of HIV

Studies have shown that the virus population early in HIV infection is homogeneous with respect to cell-tropism and nucleic acid sequence [83-851. In a few cases where the transmitting and newly infected partners have been identified, it appears that the virus transmitted represents a variant present at low frequency in the donor viral population that is non-cytopathic, macrophage tropic [84]. However, viral variability can develop rapidly after infection [83,84]. Three hypotheses have been proposed to explain the discrepancy between the heterogeneous virus population in the transmitting partner and the homogeneous virus recovered from a recently infected partner. The homogeneous virus observed in a newly infected person could reflect (a) exposure to a low titer of virus from the transmitter, (b) selective amplification of one variant after entering the new host, or (c) selective transmission of viral variants across the mucosa of the reproductive tract [84]. The observation that the transmitted virus represents a minor, non-cytopathic macrophage-tropic variant in the blood of the transmitter supports the later explanation [84]. In contrast to the reports described above, results of recent studies indicate that the biological phenotype or genotype of HIV isolated from the transmitting and newly infected partners is indistinguishable [86,87]. It was reported that individuals with rapid/high, syncytium-inducing phenotype HIV-l variants in peripheral blood often transmit this virus phenotype; individuals who are infected with rapid/high, syncytium-inducing HIV-l variants also tend to retain this virus phenotype and show rapid disease progression [85]. Evidence has also been presented for sexual transmission of multiple HIV variants with unique sequences in the gag and pol genes [87]. The discrepancy between these observations may be explained by differences in the stage of disease of the transmitting individuals, the regions of the HIV genome

sequenced, and the methods used for sequence analyses (direct sequencing from viral nucleic acids [87] or sequencing from cloned DNA [86]).

6. Anti-HIV and SIV functions of S-IgA As described above, HIV is transmitted by both homosexual and heterosexual contact; however, the current epidemic is expanding rapidly by heterosexual transmission. In both cases, the route of infection in adults is through the mucous membranes. Furthermore, in mother to infant (vertical) transmission, the gastrointestinal mucosa very likely participates in acquisition of the infection. Thus, immunity at mucosal surfaces could play a pivotal role in resisting HIV infection. Detailed understanding of the mechanisms of IgA function will be advantageous in designing an effective mucosal vaccine for HIV Here three potential mechanisms by which IgA may combat viral replication in the mucosa will be discussed: (1) extracellular neutralization that blocks HIV from infecting epithelial cells; (2) intracellular neutralization that aborts its replication once the virus has invaded epithelial cells; and (3) excretion of virus-IgA immune complexes from the LP to the luminal surface, if the virus reaches the mucosal LI? 6.1. Evidence for HIV and SW infection through mucosal epithelium

HIV positive cells have been identified in rectal epithelium of infected people [88,89] and studies in vitro have demonstrated that several intestinal epithelial cell lines are susceptible to infection by HIV [90-951. For infection of colon epithelial cells, an alternative receptor to CD4, galactosyl ceramide, is used to mediate uptake [95]. Recent studies have also shown that in SIV infected monkeys, a relatively high burden of virus was observed in the intestinal tract and positive cells were found primarily in the intestinal LP and in the GALT [%I. However, direct infection of human fetal intestinal explant cultures with HIV showed that lymphocytes and M0 are the predominant cell types infected and actively producing virus, whereas there was no

evidence for infection of the epithelial cells [97]. Besides possible direct infection of mucosal epithelial cells, HIV may adhere to the luminal membrane of the M cells and be transcytosed and delivered to underlining lymphocytes and M0 [98]. Moreover, in the postnatal transmission of HIV in colostrum and milk from mother to infant, the mucosa of the upper gastrointestinal tract, including tonsilar M cells, could also play a role [99]. Another possible route of infection for HIV is through the damaged mucosal epithelial cells allowing more direct access to the microcirculation of the rectal mucosa and infection of mucosal lymphocytes and M0. Although there is no direct evidence for infection of cervical or vaginal epithelia by HIV virus specific DNA, free-virus and cell associated virus have been demonstrated in the cervicovaginal secretions of infected women [loo-1031. An in vitro study using cell-associated virus showed that human cervical adenocarcinoma cells (ME-180) can be infected with HIV [104]. These data imply that cervical epithelium has the potential to serve as a site of HIV infection. Further, in HIV infected patients, virus specific DNA from endocervical swabs was detected in 75% of individuals infected sexually, but in only 23% of individuals infected intravenously [105]. These data suggest a significant role for surface epithelia in HIV infection and the potential importance of mucosal immunity in its prevention. In monkeys, cell-free virus can induce systemic infection from both male and female genital mucosal route of infection, and both cellfree and cell-associated virus were detected in the cervix and vagina [72-741. These studies suggest that in heterosexual transmission in both male and female, genital mucosae are primary ports of entry and dissemination of infection. 6.2. Can IgA block invasion of HIV and SIV at mucosal surfaces ? Prevention of virus infection by specific antibody (virus neutralization) has been studied in many viral systems. Classic viral neutralization is studied by mixing antibody and virus together and then assaying the amount of infectious virus by allowing the “neutralized” virus to infect

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cells. Classic neutralization mediated by IgG antibody has been studied in SIV [106-1091; and more intensively in HIV [llO-1141. However, mechanisms of neutralization mediated by IgG and IgA are strikingly different. IgA is generally thought to block binding to host cells while IgG exhibits a variety of functions from preventing attachment to inhibiting viral transcription. Classic neutralization of influenza virus infection by IgG and IgA has been extensively studied [11.51181. The mechanism(s) of classic neutralization are dependent on the virus studied, the isotype of the antibody and even on the cell type in which the neutralized virus is assayed. The main function of S-IgA in host defense is generally believed to be an immune barrier to block the adherence of microbes to mucosal epithelial cells [119-1241. IgA mediated protection of the host has been described in viral infections of the respiratory tract [125-1291. Increased levels of specific S-IgA antibody have been shown to correspond to a decrease of viral shedding as well as severity of disease for many respiratory viruses. It has also been demonstrated that S-IgA antibody decreases the secretion of intestinal viruses such as rotavirus and poliovirus [130,131]. In addition to the direct correlation between levels of S-IgA and degree of virus shedding, it has also been shown with anti-Herpes simplex virus S-IgA antibody that the duration of virus shedding was shortened [132]. IgA antibodies administered passively to mucous membranes have been shown in several experimental systems to be capable of inhibiting microbial infections [133-1371. Finally, specific HIV antibody at the genital mucosa correlates with lower quantities of free virus found in cervico-vaginal secretions of infected women [103]. Thus, it has been extensively demonstrated that S-IgA at the mucosal surfaces can play a significant role in decreasing virus shedding and in preventing viral entry into epithelial cells. 6.3. Intraepithelial cell neutralization of viruses by S-ZgA

Besides the traditional function of S-IgA as a mucosal barrier, it has recently been demonstrated that monoclonal IgA antibody to the HN

2.3-51

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envelope glycoprotein of Sendai virus and to the HA glycoprotein of influenza virus can neutralize the virus intracellularly [138,139]. In this experimental system, polarizable epithelial cell monolayers were grown in tissue culture chambers on top of a permeable nitrocellulose filter. The cells used for these experiments are a line of MDCK cells transfected so as to express the rabbit polymeric immunoglobulin receptor (pIgR) [140]. The virus was added to the top of the monolayer and dimeric IgA antibody was added from the lower chamber. For both Sendai and influenza virus, polymeric IgA (pIgA) antibodies, but neither specific monomeric IgA antibody, nonspecific polymeric IgA antibodies, nor specific IgG antibodies, could reduce virus titers [138,139]. This intraepithelial cell neutralization of virus occurs during the transcytosis of IgA from the basolateral to the apical surface, i.e., by the normal route by which mucosal pIgA reaches the external secretions. Based on the Sendai and influenza virus results of intracellular neutralization, it is logical to assume that intracellular neutralization could occur with IgA antibody specific for gp 120/130 of HIV and SIV There are no data yet to indicate that IgA intracellular neutralization will occur with antibody against internal viral proteins as opposed to the envelope viral proteins. In the case of HIV and SIV, the major core antigen, gag, reverse transcriptase and integrase are suitable targets for IgA antibody to disrupt function and block viral replication. If IgA is able to interact with the reverse transcriptase inside the cell, synthesis of proviral DNA may be aborted. This could abort viral infection at a stage before integration of the viral DNA into the host chromosome. 6.4. Dimeric ZgA mediated excretion of virus through the epithelium Another novel function of mucosal IgA that has been described is an excretory function [141]. With the same pIgR-expressing MDCK cells mentioned above, it was shown that in addition to transcytosing dimeric IgA, these cells will also transcytose immune complexes of anti-DNP (dinitrophenyl) IgA and the model antigen DNP-

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bovine serum albumin when the soluble immune complexes were placed below the polarized epithelial monolayer [141]. The excretion of immune complexes was further extended to show that mixed immune complexes between multivalent antigen and a mixture of pIgA and monomeric IgA or IgG antibodies are also transported from the basolateral to the apical surface [142]. It is likely that local transcytosis of IgA immune complexes from the LP occurs in vivo in the mucosal membranes. The upper size limit of these transcytosed immune complexes or whether IgA might mediate this exocytic process for viable or inactivated pathogenic microorganisms of the size of viruses has not been tested. The transcytosis of inert complexes, however, leads us to hypothesize that virus located near the basolateral surface of mucosal epithelial cells might be eliminated by IgA mediated endocytosis and transcytosis. This could be significant in light of the data showing that a high level of virus was found in the LP of the intestinal tract of SIV infected monkeys in the end-stage of disease, [96]. Furthermore, in HIV infected patients, virus RNA was detected in the LP of the gut [143]. These results suggest that in HIV and SIV infections the lower gut lining may be both a portal of initial infection and a target of disseminated infection. Thus, IgA-mediated excretion of HIV and SIV from the LP could reduce the viral load and decrease dissemination of virus.

mediating transcytosis of dimeric IgA from the basolateral to the apical surface. Furthermore, internalized Sendai virus-specific IgA mAb was able to neutralize Sendai virus intracellularly after virus was introduced from the apical surface and IgA from the basolateral surface. When the pIgR expressing Vero cells were transfected with SIV proviral DNA, infectious virus was subsequently released. As early as 18 h post transfection, the supernatant was able to infect CEM X 174 cells, as shown by syncytia formation and immunofluorescent staining with anti-gag mAb. At day four post transfection, the released infectious virus peaked at a TCID of 103/ml and slowly declined, as monitored by its ability to infect CEM X 174 cells. When the transfected Vero cells were immunostained with anti-gag mAb, about 5% of cells were positive. The expression of the transfected proviral DNA at the RNA level was determined by a more sensitive in situ hybridization protocol which reached about 60% of positive cells. With this model system, we are prepared to investigate intraepithelial cell neutralization and excretion of SIV by IgA antibody. Results generated from these studies may identify a single mAb or a set of IgA mAbs with potent anti-viral properties from which the reacting epitope(s) can be mapped. This information can subsequently be used for designing a mucosal vaccine.

6.5. Model system for neutralization of SN by

mucosal

7. New developments

for HIV and SIV specific

vaccines

W

For an in vitro model of function of IgA, three factors are required, i.e., the cells (1) should be capable of forming a polarized tight junction monolayer; (2) must be able to transcytose IgA; and (3) must be able to support SIV replication. In order to satisfy these requirements, we have begun to develop an in vitro model system for studying intracellular neutralization and excretion of SIV by IgA antibody. Human polymeric immunoglobulin receptor gene cloned into a eucaryotic expression vector was transfected into polarizable primate epithelial cells (Vero C 1008). The expressed receptor was functional in

The sexual transmission of HIV has become the main route of infection in the past several years, and AIDS is now the most threatening STD in the world (see section 1). The rapid spread of the pandemic calls for the international vaccine research community to apply the most advanced technology and knowledge to the development of effective HIV prophylactics. An ideal vaccine for the prevention of HIV infection should effect primary protection at the mucosal site of entry and secondary humoral and cell mediated immune protection from systemic spread. In addition, such a vaccine should have the ease of administration, distribution and pro-

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duction so that world wide immunization is practical. To this end, we have briefly summarized our recent progress for the understanding of induction of HIV and SIV specific immune responses by mucosal immunization and its possible application for the development of an HIV and SIV specific mucosal vaccine. 7.1. Novel mucosal vaccine delivery vectors

Several types of antigen delivery systems are currently being developed and include: (1) live attenuated bacterial vectors, (2) biodegradable microspheres and (3) DNA. In this section, we have summarized progress on these three different antigen delivery systems for the induction of HIV and SIV-specific mucosal and peripheral immune responses. 7.1.1. Live attenuated bacterial vectors To infect humans, many microbes have adapted an ability to either replicate at the mucosal surface or disseminate to establish systemic infections. Following infection, the host develops both mucosal and systemic protective immune responses to clear the infectious agents from the system and to prevent future infections. Genetic engineering makes it possible to modify a microbe of choice so that it is no longer pathogenic to humans while retaining its ability to replicate at the mucosal surface. Not only does this procedure create a genetically attenuated live vaccine against a single pathogen, it also provides a live vaccine vehicle with the capacity of delivering foreign antigens to a human host. Ideally, such a recombinant vector would deliver HIV-l antigens orally in a single dose and would confer both peripheral and mucosal protective immunity to the virus. Current efforts to develop live vaccine delivery vehicles have concentrated either on viral vectors such as attenuated vaccinia virus, poliovirus and adenovirus or on bacterial vectors such as BCG and Salmonella species. While a viral vector is relatively easier to manipulate, the main concern in using a live viral vector is safety. The lack of viricidal agents makes it difficult to arrest generalized infections in an immunocompromised human host. The advantage of using a live

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bacterial vector, on the other hand, is the availability of a wide variety of antibiotics should a breakthrough of virulence occur in an immunocompromised individual. However, the method of using traditional attenuated bacteria such as BCG or Salmonella typhi as vaccine vectors suffers from the inherent invasive nature of the bacteria which persist too long in the human host or cause bacteremia. Thus, genetically engineered bacterial vectors with stable and defined deletion of virulence genes would be preferred for use in humans. Vibrio cholerae is the causative agent of the diarrhea1 disease cholera. Vibrios enter the human host through ingestion of contaminated food or water, adhere to the mucosal membrane of the small intestine, and colonize the gut for about 1 week. The principal virulence factor which causes the disease symptoms such as vomiting, fever, and watery diarrhea has been identified as cholera toxin (CT). Secreted by the bacteria as an enterotoxin, CT enters the intestinal epithelial cells, activates the host cell’s adenyate cyclase complex, and results in cellular dehydration. Recent studies have deduced the molecular structure of CT. A functional CT molecule has two different subunits, CT-A and CT-B. The CT-A subunit is composed of a polypeptide with two domains, CT-Al and CTA2, which are associated by a disulfide bond. It is the CT-Al that is responsible for the toxic effects of the toxin [144,145]. A recent study shows that the CT-A2 polypeptide orients the A subunit with B subunits and is required for the formation of the cholera holotoxoid, which has one CT-A subunit inserted into a CT-B pentameter [146]. The CT-B pentameter is highly immunogenic and binds to the membrane ganglioside, GMl, of host cells. The studies described above led the Mekalanos group at Harvard University to develop a new generation of live cholera vaccines in which the CT genes are deleted from the bacterial genome and the CT-B gene is re-introduced by genetic engineering. Moreover, these genetically attenuated strains harbor a further deletion within the recA gene and are rendered incapable of genetic recombination and thus are essentially not capable of reversion. Virus Re-

search Institute (VRI), in collaboration with the IJS Army’s Walter Reed Research Institute, has tested sevei new vaccine candidates in human clinical trials for safety and efficacy evaluation. The results indicate that one such strain, Peru3. is safe. immunogenic and confers protection against a virulent cholera challenge 11471. As the infection of Vibrio cholerue elicits high levels of mucosal and systemic immune responses, Peru3 may become a novel vaccine vector for general utility and especially for new oral prophylactic HIV vaccines that elicit both mucosal and systemic immunity. In fact, VRI has genetically engineered a live attenuated V. cholerae to express HIV-l antigen in the form of non-toxic recombinant holotoxin. As illustrated in Fig. 3, a fragment of HIV envelope gene replaces the toxic CT-Al subunit. The reconstituted toxin genes are manipulated into the recA locus of the vaccine strain Peru2. This construct enables the bacteria to stably secrete the HIV-l antigen during their replication at the mucosal surface. The parental strain Peru2 was generated by removing all the DNA sequences that correspond to the ctx locus through double homologous recombination between the chromosomal DNA of wild type Vibrio cholerue C6709 (CDC) and the shuttle plasmid pAR60 (J. Mekalanos). The genetic manipulation of foreign genes into the chromosome of bacteria is also achieved by homologous recombination between the DNA sequences present both in the chromo-

Cholera-HIV

7

holotoxln

Fig 3. Induction of antigen-specitic immune responses by rccomhinant HIV antigen.

mucosal and q%cmic V. cho/m7c~ expressing

some and on the shuttle plasmid. Consequently, a double recombinant event results in a site specific insertion of the foreign sequences and a coincidental deletion of a specific bacterial locus. As the shuttle plasmid is unable to replicate within Vibrio cholerae without being integrated into the chromosome, the recombinant bacteria are selected by a two step procedure. Firstly, the bacteria that acquire the shuttle plasmid sequences through a single recombination event, merodiploid, are selected by utilizing a marker encoded by the shuttle plasmid. This selectable marker is positioned in the plasmid such that its subsequent loss identifies the bacteria in which a single recombinant has condensed the final double recombinant structure. Currently, VRI is testing the immunogenicity of this oral HIV-l vaccine candidate in rabbits. Salmonella typhi, the other human pathogen being proposed as a vaccine vector, is the causative agent of typhoid fever in humans. Like Vibrio cholerae. Salmonella typhi enters the human body via contaminated food or water. Unlike Vibrio cholerae, Salmonella do not secrete any known toxins and exhibit a tissue invasive phenotype. Once the bacteria invade the intestinal mucosa, they multiply and survive even after being taken up into the MO phagolysosome. It is this distinct ability to survive inside the MO that contributes to the pathogenicity of Salmonella [148,149]. Recently, Dr. Sam Miller at Massachusetts General Hospital (MGH) has identified a two component regulatory system, phoP, phoQ and virulence genes that play an important role in the bacteria’s ability to survive in the MO phagolysosome. Two genes, pagC and pagD, phoP activated gene C and D, are transcriptionally activated lOO-fold once the bacteria enter the MO [1.50]. Thus, it provides an in vitro inducible promoter for the expression of a foreign antigen from within antigen presenting MO. As Salmonella also colonizes the human gut mucosa and stimulates a strong mucosal immune response and cell-mediated immune responses, the new attenuated non-invasive strain may become another novel vaccine vector for the development of an oral HIV-l vaccine. VRI has made a number of Salmonella-HIV constructs in which HIV-l gag, nef and envelope genes are

4%

Salmonella-HIV

Cell Mediated Immune FkZSpOllWS

Fig. 4. Application of recombinant antigen for mucosal immunization CM1 responses.

S. ryphi expressing HIV induces antigen-specific

introduced into Salmonella chromosomal DNA to replace the deleted pagC gene (Fig. 4). Such a construct has the potential to stimulate cell-mediated immune responses to destroy cell associated HIV-l virus. In order to test the immunogenicity of these strains in mice, Salmonella typhimurium, instead of Salmonella typhi, is used as the vaccine vector. VRI, in collaboration with MGH, is evaluating the immunogenicity of these HIV-l vaccine candidates. 7.1.2. Biodegradable

microspheres

Microencapsulation technology has a long history of usage in drug delivery. In recent years, vaccine researchers became interested in using the technology for antigen delivery. The attraction comes from the following rationale. Today’s biotech industry is capable of large scale production of recombinant subunit antigens lead this to the development of the lucrative HBV vaccine. However, many recombinant subunit antigens contain labile epitopes and are poorly immunogenic. Therefore, the successful vaccine formulation of these antigens requires both a delivery vehicle and a safe immune stimulant. The enthusiasm for microencapsulation also results from studies of the induction of mucosal immunity by microbes. The majority of infectious agents utilize mucosal surfaces as the primary portal of entry. As a protection mechanism, the host develops both systemic and mucosal protec-

tive immune responses against the infectious agent. In this process, the structure of PP plays an important role in taking up infectious pathogens to pass them to the APC. If one could mimic this antigen stimulation by microencapsulation of a recombinant antigen having the size and biophysical properties required for the uptake by the PP, one would be able to make a new generation of recombinant subunit vaccines. In the past few years, the group of investigators from the University of Alabama at Birmingham (UAB) and the Southern Research Institute (SRI) have become pioneers in exploiting this new technology. In their studies, a biodegradable and biocompatible polyester of poly DL lactide-coglycolide (PLGA), which has a long history of safe usage in drug delivery, is used to microencapsulate immunogens. As these microcapsules protect antigens through the GI tract and then release them to the mucosal surfaces, it significantly improves the efficiency of oral delivery of the antigens. In addition, the microencapsules release antigen for sustained periods of time, resulting in enhanced stimulation of mucosal and systemic immunity [151]. Furthermore, the UAB and SRI team demonstrated that the entrapment of antigens in PLGA microspheres of l-10 microns in diameter appears to have a remarkable effect on the uptake on the antigens by the M cells in PP [151]. However, one problem with PLGA system is that it has to form microspheres in the presence of organic solvents such as ethyl acetate. Most of immunogens, on the other hand, are dissolved or prepared in aqueous solutions. The PLGA microencapsulation process is essentially a waterin-oil emulsion by high speed stirring. While this process has worked for microencapsulation of peptide based drugs and immunogens like staphylococcal enterotoxin B [152], it is very difficult to preserve the immunogenicity of large protein antigens, especially those possessing the conformation dependent epitopes on labile immunogens such as enveloped viruses. In fact, this physical property of PLGA is the main obstacle for microencapsulation of many vaccine candidates. The research group at VRI is working on an alternative microencapsulation process using a

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different biodegradable polymer, polyphosphazene. Unlike PLGA, polyphosphazene is water soluble and ion cross-linkable [153]. As the gelation of polyphosphazenes by ionic crosslinking occurs in aqueous solution at room temperature without high speed stirring, it becomes possible to preserve conformationally sensitive epitopes on labile immunogens during the microencapsulation process. VRI has developed a procedure to produce antigen bearing microspheres of desired size [154]. In this system, several parameters determine the kinetics of antigen release; the polyphosphazene concentration controls the amount of antigen presented in the microspheres; the polyphosphazene side chain determines the interaction of the polymer with a particular antigen and finally, polyphosphazene microspheres coated with poly( L-lysine) determines the time course of antigen release from the polyphophazenes matrix. Currently, VRI is working on microencapsulation of recombinant HIV gp120 antigen with polyphosphazenes as an oral HIV vaccine candidate which may elicit both mucosal and systemic immune responses.

7.1.3. Non-replicating HIP1 particles The advantages of the above described bacterial vaccine vectors are the cost, ease and number of administrations and the regulated expression of many heterologous genes of recombinant gp120. The main limitation of these live vectors could be the nature of the heterogous antigen presented to the immune system For example, the HIV antigen synthesized by bacterial vectors lacks all the post-translational modifications that native HIV antigens possess. It is unclear whether these recombinant antigens will stimulate protective immunity. The alternative is to deliver in polyphosphazene recombinant HIV-l gp120 produced from eukaryotic cells. However, the ability of this subunit antigen alone to elicit protective immunity remains a question. Therefore, it may be necessary to produce HIV antigen complexes that are more like those found in the native virus particles. The fact that HIV-l gp120 presented in a virus like particle by vaccinia virus vector is a more potent immunogen than soluble

gp120 supports this hypothesis. Even in these recombinant virus like particles, the HIV-l envelope protein is still different from the native gp120 in the HIV-l virions. It is possible that other viral antigens may be required for the induction of protective immunity, or that multiple HIV-l gp120 plus other viral or cellular antigens are required. It is also possible that virus replication is a prerequisite for the effective antigen presentation, which leads to the concept that a live attenuated HIV-l virus may be the ultimate solution for HIV-l vaccination. As HIV1 is a human retrovirus, which integrates its genome into the chromosomal DNA of a host to complete its replication cycle, a live attenuated HIV-l vaccine will face a great deal of safety concerns. We propose to make a recombinant HIV-l virus that is like a native virion, but non-replicative. The long terminal repeats (LTR) of HIV1 provirus’ have many functions necessary for virus replication. The LTRs contain the transcriptional promoter and terminator for the expression of all viral genes. They also contain sequences recognized by HIV-l integrase which inserts the virus genome into the host chromosome. As the two repeated sequences at both ends of HIV-l RNA genome derive from the LTRs, they are required for HIV-l reverse transcriptase (RT) to convert the RNA genome into a DNA provirus. VRI’s research group has made a recombinant HIV-l provirus in which the 5’ LTR is replaced by the CMV promoter and the 3’ LTR is replaced by the poly A signal from HSV tk gene. We demonstrated that the recombinant HIV-l provirus is capable of producing all the viral proteins and assembling virus particles. Once the virus enters a host cell, the HIV-l RT is unable to convert the RNA genome into the DNA provirus. Therefore, the HIV-l virions produced by the recombinant provirus appear like a native non-replicative HIV-l viron. VRI has introduced this recombinant HIV-l provirus into an established mammalian cell line to make a stable viron producer for HIV-l vaccination. The combination of this virus antigen with polyphosphazenes microencapsulation may provide a more effective oral formulation for an HIV-l vaccine.

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vaccine for HZV

An important part of the rationale for using a live attenuated virus is that the viral proteins synthesized during the virus replication are immunologically more potent than the antigens taken by the cells. In order to mimic this part of virus replication without using live virus, VRI is testing the method of direct DNA immunization. In the past 2 years, many studies demonstrate that intramuscular injection of naked plasmid DNA containing an antigen gene results in efficient expression of the antigen in the tissues surrounding the injection site. Thereafter, both humoral and cell mediated immune responses are elicited against the input antigen. We propose to use the non-replicative HIV-l provirus DNA as a vaccine. A cell that expresses all the virus proteins encoded in the plasmid should look like an HIV-l infected cell and produces nonreplicative virus like particles. If indeed the intracellular expression of virus antigens, or the assembling of virus particles in the membrane of infected cells, are required to stimulate effective protection against HIV-l infection, this approach will be a safer alternative to the usage of live attenuated virus. There are several potential methods to apply DNA vaccines for the induction of mucosal immunity. One of them is to use polyphophazenes to microencapsulate plasmid DNA to deliver the vaccine to the mucosal membrane. Previous studies have shown that a successful DNA immunization often needs multiple injections of the DNA plasmid. The release of DNA molecules from polyphosphazene microspheres should last for a sustained period of time, reducing the number of doses administered. As the uptake of particulate material by M cells of PP and subepithelial M0 is limited to particles which have diameters of 10 microns or less, microencapsulated DNA may be used orally, intranasally, or intrarectally. It is anticipated that an effective HIV-l vaccine may be a combination of different formulations. For example, it may be a cholera-HIV or salmonella-HIV primary immunization followed by a gpl20/polyphosphazenes boost. A primary oral or intrarectal immunization of non-replicative HIV-l DNA or viron may combine with an

41

intrarectal or oral boost with gp120 microspheres. VRI is testing every combination of the above described methods in animals in order to define an optimal approach. In summary, we are devoted to developing several novel mucosal vaccine delivery vectors in an attempt to stop the spread of one of the most devastating human infectious diseases. The success of this new vaccine vector research will not only help to make an effective HIV-l vaccine, but will also result in a new generation of vaccines delivery vehicles to improve the quality of human life. 7.2. Induction of SZV-specific immune responses in the genital, rectal and urinary tracts Immunization via the mucosal associated lymphoid tissue might be expected to elicit S-IgA antibodies in the genito-urinary and lower digestive tracts [17,155]. Indeed, oral immunization in rodents induced antibodies in the genital tract, and adoptively transferred murine mesenteric lymph node cells home to the genital tissues [155]. Direct administration of SRBC into PP of Sprague-Dawley rats induced vaginal IgA and IgG antibodies [156,157]. These experiments suggest that sensitized B cells may home from GALT to the genital tract. However, oral immunization in non-human primates with the recombinant SIV gag p27 fused to the yeast retotransposon virus like particles (p27:Ty VLP) failed to induce significant S-IgA antibodies in the genital or rectal tract [158]. Similarly, microencapsulated formalin treated SIV administered orally to rhesus macaques failed to elicit vaginal antibodies or protection when challenged with live SIV by the vaginal route [159]. However, augmenting rectal, vaginal or male urethral administration followed by oral immunization elicits S-IgA and IgG antibodies and T cell responses [160-1621. Furthermore, augmenting oral (or intra-tracheal) by prior IM immunization with SIV induced vaginal IgG and IgA antibodies to SIV and 5 out of 6 macaques were protected when challenged by the vaginal route [159]. A subcutaneous route of immunization was developed recently in non-human primates

[targeted lymph node (TLN ) immunization]. which targets the genito-urinary-recta1 associated lymphoid tissue [.59]. S-IgA and IgG antibodies were elicited in the vaginal, male urethral, rectal and seminal fluids, urine and serum. CD4 T cell proliferative responses to p27 were elicited predominantly in the targeted internal iliac. as well as the inferior mesenteric lymph nodes and the spleen, but not in the unrelated lymph nodes.

7.2.1. Compurutive yuantitutiw andysis o,t p-77,spc~$c IgA rrntihodic.v follolvillcq ,fivc routes of immunizrrtiorr As mucosal immune rcsponscs can he achieved by direct mucosal and indirect TLN routes of immunization. it seemed important to compare the immune responses elicited by direct vaginal, male genito-urinary or rectal mucosal with the subcutaneous TLN route of immuniza-tion (591. The highest proportion of p27-specitic IgA antibodies were found in the sera of male and female macaques immunized by the TLN route (3.3-3.9%), and these were significantly greater (p < 0.05) than those after recta-oral. male genito-oral or intramuscular route of immunization ( 1.9-2.X% : Table 1). Rectal washings showed a similar proportion of p27 specific IgA antibodies after TLN ( 1.7 z 0.24% ) as those following mucosal immunizations ( 1.6 1.7% ). with the exception of recta-oral immunization (1.2 t- 0.26% ). The proportion of vaginal p27specific IgA antibodies was significantly higher

Koute

Numbers 01

ot

I‘LN

___

1’77 ywcilic

IsA

in total IpA ol dillcrent

Ruids

anlmill\

mununi/.ation

__-

‘IS, 01 SI\’

( 17c.’ 0.02) after TLN (3.8 +- 0.38%) than that following vagino-oral immunization (2.3 -+ 0.28% ). Similarly, seminal fluid p27-specific IgA antibodies were higher after TLN (3.3 ? 0.15%) than male genito-oral immunization (1.4%), but the number of samples was inadequate for statistical analysis. Urinary and urethral p27specific IgA antibodies were comparable with those following male genito-oral immunization. Intramuscular immunization failed to elicit any mucosal. urinary or seminal fuid p27-specific 1gA antibodies (Table 1). Examination of the proportion of IgA antibodies to p27 in each fluid after TLN immunization with the recombinant SIV p27:Ty-VLP and aluminum hydroxide suggests the following. (a) Genital, urinary. rectal, seminal fluid and serum IgA and IgG antibodies were elicited. (b) Vaginal, urethral. rectal fluid and urinary IgA antibodies to p27 are polymeric (with J chains) and secretory (with SC). (c) Higher concentrations of vaginal, rectal, urinary and serum IgA antibodies arc achieved after TLN than after vagino-oral immunization. (d) IgA antibody concentrations in serum were higher, in rectal fluid and urine they were similar, and in seminal and urethral fluid they were slightly lower after TLN than those found after male genito-oral immunization. ce) Serum and rectal IgA antibodies to p27 showed higher concentrations after TLN than recta-oral immunization, and only the former elicited detectable antibodies in the urine and urethral washings.

-_

___ 5( ?)

‘I‘LN

4

i I

Vagio-Oral-IM

51

I

(ienito-Oral-IM Kecto-Oral-IM

4 ‘I h( I)

IV

51’

__~

)

+J

KcCt:ll __-_ 1.7

Vapm;d

I .(I

ih

I.7

7 :

I’rethral

Seminal

I).(1

1.3

0

0

I .f,

0.‘)

I .J

I.2 (I

I)

.._

I!rine

Serum

0.x

3..3

0.7

4.0 3.0

1).X

2.0

0

0

2.x

0

0

2.1

H. Kiyono et al. I Advanced

Drug Delivery Reviews 18 (199-T) 2.3-51

7.2.2. Comparative proliferative responses of T lymphocytes following jive routes of immunization

TLN immunization elicited a similar pattern of responses to p27:Ty-VLP in the lymph nodes as the 3 mucosal routes; internal iliac, inferior mesenteric and iliac-paraortic, in addition to the splenic and circulating T lymphocytes. These 4 routes of immunization differed from the IM route, in which only the splenic and circulating T cells and none of the lymph node cells responded to ~27. The magnitude of T cell proliferation after TLN immunization was similar to that found after genito-oral immunization in females, but lower than those after genito- or recta-oral immunization in males. A comparison of the lymphoproliferative responses after TLN with those of the three mucosal routes of immunization showed no significant difference in the distribution of lymph node cells sensitized to the p27 antigen. The dividing T cells belonged to the CD4’ subset, irrespective of the route of immunization [160-1621. It is interesting that the magnitude of T cell proliferation after TLN immunization was similar to that found after female genito-oral but lower than those after male genito-oral or recta-oral immunization. TLN, like the mucosal routes differed from IM immunization by sensitization of CD4’ T cells not only in the circulation and the spleen but also in the internal iliac. inferior mesenteric

Table 2 Analysis of antigen-specific IgA immune responses immunized rhesus macaques with p27:Ty-VLP Route of immunization

Numbers animals

of

in cultures

Source

and the common iliac-para-aortic lymph nodes [59]. Localization of specific T and B cells to the primary draining lymph nodes of the genitourinary and rectal tracts might be essential in preventing transmission of HIV by infected Langerhans, DC or M0 from the mucosal tissues to these lymph nodes. The T and B cell functions might prevent viral latency and producing a viral reservoir in the primary draining lymph nodes, if the mucosal immune barrier were to be breached. The significance of this observation has been greatly enhanced recently by the evidence that HIV is found early in infection in the lymph nodes, and that the virus remains latent probably in the follicular DC L163.1641.A central immune barrier is also induced, which is comparable to that found after IM immunization, in that splenic and circulating proliferative and helper T cells, as well as sensitized B cells, IgG and IgA antibodies are found. 7.2.3. Comparative CD4’ Th helper function in B cell antibody synthesis following five routes of immunization Reconstituted splenic CD4’ T cells, B cells and M0 showed consistently higher p27-specific IgG than IgA antibody synthesis after all but the recta-oral route of immunization, where the levels were equally low (Table 2). In contrast, reconstituted iliac lymph node cells yielded comparable p27-specific IgA and IgG antibodies

containing

CD4 ’ T cells, B cells and M@ isolated

of cells and isotype

Adjuvants

43

of p27 specific

antibodies

Ileum

from mucosally

(absorbance

at 429 nm)

Spleen

IgA

IgG

IgA

IgG

TLN TLN

38) 39)

Al(OH), AI(

0.4 0.7

0.7 0.6

0.3 0.4

0.8 I.0

Genito-Oral Genito-Oral Reck-Oral

3(P) 3( 6 ) x 6 )

CT-B CT-B CT-B

0.3 0.6 0.2

0.2 0.5 0

0.2 0.1 0.2

0.4 0.2 0.2

IM

3(6/Y)

Al(OH),

0

0

0.3

0.5

44

H. Kiyono et al.

I Advanced Drug Delivery Reviews 18 (1995) 23-51

after TLN, higher IgA than IgG antibodies after any of the three mucosal and no antibodies after the IM route of immunization (Table 2). Reconstitution experiments with enriched CD4+ T cells, B cells and M0, separated from the SP and iliac lymph nodes after TLN immunization elicited SIV p27 specific IgA and IgG antibodies. However, whereas splenic cells elicited higher IgG than IgA antibodies, the iliac lymph node cells yielded comparable IgG and IgA antibodies. A comparison of TLN with the three mucosal and the IM routes of immunization showed similar results with reconstituted SP cells, in that p27-specific IgG antibodies were higher than the IgA antibodies. However, reconstituted iliac lymph node cells yielded higher IgA than IgG antibodies after the three mucosal routes of immunization, similar IgA and IgG antibodies after TLN and no antibodies after IM immunization. It appears that immunization by the TLN route elicits regional and central CD4’ Th cells and B cells, as was found in genito-oral or recta-oral immunization, but Th helper function was higher especially for IgG antibodies. Surprisingly, p27-specific IgG antibody synthesis by splenic cells was higher than those found after IM immunization. The subcutaneous TLN route of immunization combines most of the advantages of mucosal and IM immunization, in generating local mucosal, regional lymph node, and central splenic and circulating immunity. Furthermore, the alternative TLN route of immunization achieves mucosal immunity with two to three injections, as compared with five mucosal applications; this reduces significantly the experimental time and expense in non-human primate experiments. This route of immunization may facilitate investigations of potential vaccines against HIV and other STD, as broadly based and consistent mucosal immune responses are induced. The adjuvant used is aluminum hydroxide, which has a long safety record in humans. TLN immunization may also facilitate compliance and allow flexibility in administration of a vaccine in different countries, a sub-cutaneous immunization might be more acceptable to some than genital or rectal immunization. Other STD, which are open to this approach are human papilloma virus, herpes

simplex, candida, chlyamidia or gonococcal infection. The functional significance of the four levels of immunity will need to be tested by mucosal challenge with SIV or other microbial agents. 7.3. Mucosal TLN immunization induced SIVspecific Thl and Th2 cells for antigen-specific IgG and ZgA B cell responses In AIDS, the dichotomy of Thl and Th2 predominance in HIV-infected individuals has recently become a central issue for debate. It was originally demonstrated that a switch from the Thl to Th2 responses occurs during the development of the HIV infection [165]. As a result, the measurement of IL-2, IL-4, and IL-10 following in vitro stimulation with recall antigen or T cell mitogen from different stages of patients with the HIV infection resulted in the reduction of IL-2 synthesis (Thl) and the increase of IL-4 and IL-10 production of (Th2) with disease progression. However, recent studies revealed that a shift from Thl to Th2 responses do not occur during the progression of HIV infection [166,167]. Further, it was also demonstrated that HIV preferentially replicates in CD4+ T cells with phenotypes of ThO and Th2 cytokine synthesis [167]. When we consider the development of a mucosal vaccine which can induce HIV and SIV-specific protective immunity in both mucosal and systemic compartments, we must also account for the dysregulation of Thl and Th2 cell profiles in AIDS. Through our CMIG efforts, we have initiated experiments characterizing antigen-specific Thl and Th2 cytokine profiles with B cell responses in rhesus macaques mucosally immunized with inactivated whole SIV or a combination of gp120 and ~27. When two groups of rhesus macaques (five monkeys per group) were immunized via the genito-urinary-recta1 lymphoid tissues (TLN immunization), antigen-specific B cell responses of IgM and IgG isotype were detected in peripheral blood mononuclear cells (PBMC) after the secondary immunization. Further, following tertiary immunization, higher numbers of SIV-specific IgG SFC were observed. These findings suggested that TLN immunization elicits

H. Kiyono et al. I Advanced

Drug Delivery Reviews 18 (1995) 23-51

SIV-specific IgM and IgG antibody producing cells. With regard to antigen-specific IgA B cells, small numbers of antigen-specific IgA B cell responses were noted. The increased numbers of SIV-specific IgA antibody producing cells were induced following the third immunization. Thus, three monkeys demonstrated good IgA B cell responses. These results further suggested that TLN immunization is a useful route for the induction of antigen-specific IgA B cell responses in addition to IgM and IgG antibodies. To study the induction of Thl and Th2 type CD4+ T cell responses by TLN immunization with SIV, IFN-y an IL-4 specific ELISPOT assays were employed. Although we could not detect both IFN-y and IL-4 producing cells in the CD4+ T cells isolated from PBMC of SIV-immunized monkeys after the primary immunization, it was interesting to note that a higher frequency of IFN-y producing Thl type cells were induced after the secondary immunization in comparison to the number IL-4 producing Th2 type cells. Thus, the ratio of Thl to Th2 was greater than 1.0. In contrast, the increase of IL-4 secreting cells was seen in CD4+ T cells isolated from PBMC of rhesus macaques which received the tertiary immunization. To this end, the ratio of Thl:Th2 became less than 1.0 in most of the immunized monkeys. The results obtained by T and B cell analysis suggest interesting patterns of the relationship between Thl:Th2 cell ratios with the isotype of B cell response. When SIV-specific IgM and IgG B cell responses were induced by the TLN immunization, the ratio of Thl and Th2 was above 1.0. The ratio was approximately 2.0-3.0 in four monkeys which possess high SIV-specific IgG responses. Thus, dominant Thl-type responses may contribute to the initiation of antigenspecific IgG responses in TLN immunized primates. Further, it was also important to indicate that when the ratio of Thl and Th2 shifted to a Th2 dominant type, antigen-specific IgA B cell responses were induced. Thus, three monkeys which had high SIV-specific IgA B cells showed a ratio of Thl and Th2 which became less than 1.0. Indeed, the range of this Thl:Th2 ratio was 0.40.8. In contrast, primates which did not have

45

detectable levels of antigen-specific IgA B cells, the Thl:Th2 ratio was greater than 1.0, with the exception of one monkey. Therefore, the induction of an antigen-specific Th2 type response might be an essential element in the generation of SIV-specific IgA B cell responses.

Acknowledgments This work is supported by CMIG for AIDS AI 35932, AI 35545 and AI 35547 and AI 26449 and HL 37117 from NIH. We thank Ms. Sheila Shaw for typing this article. We also acknowledge the helpful advice from the DAIDS branch of NIAID, and also especially Dr. Bonnie Mathieson.

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