The immune response to HIV

Chapter

|4|

The immune response to HIV Clive M. Gray, Pamela Gumbi, Lycias Zembe, Mopo Radebe, Bruce Walker

INTRODUCTION Most people in the world live in poverty-stricken conditions where they are continuously confronted with a plethora of pathogenic organisms—some successfully repelled, some resulting in clinically overt disease, and others resulting in persistent latent infection. The human immune system has evolved to combat these genetically diverse organisms, including viruses, bacteria, and protozoa, through genetically governed responses involving multiple receptors and ligands. Even with clinically overt or persistent infections, most people with an intact immune system ultimately survive most infections. In marked contrast stands HIV infection. The spread of HIV-1 worldwide represents one of the great challenges to confront host immunity, since the key target is the CD4 T cell lymphocyte, infection and depletion of which severely undermines effective immune responses. As a result, most people who become infected and remain untreated will ultimately succumb to one or more of the large variety of infectious organisms that humans are confronted with on a daily basis. By 2010, there was an estimated 2.4–2.9 million people becoming newly infected with HIV, with 1.8 million dying of AIDS-related causes (http://www.unaids.org, 2011 Global Report). The ultimate solution to the HIV epidemic relies on the development of an effective vaccine that can be delivered to those at risk. The ability to achieve this elusive goal will be facilitated by a comprehensive understanding of the key immune responses that contribute to protection from infection, or protection from disease progression in those who become infected. In this chapter, we will review the current state of knowledge of what is needed for an AIDS vaccine, first by comparison to effective immune clearance

of acute viral infections (such as influenza, rotavirus, or respiratory syncytial virus) as well as acute viral infections followed by latent infection (herpes simplex virus or Epstein–Barr virus). This will allow a foundation for discussing why successful immune mechanisms are not functional in the majority of HIV-1-infected individuals. Much knowledge on the first immune events during acute HIV-1 infection has also accumulated, providing additional clues to the arms of immunity that are triggered upon first encounter with the virus. The field has been shaped by the hypothesis that the initial immune response to HIV infection is translatable to what would be expected or required from a vaccine. We will discuss clues as to what may constitute a protective immune response from disease progression in a small proportion of people who are HIV-1 infected, but can spontaneously contain viral replication to only a few RNA copies. Finally, we will discuss some of the failures and partial successes of recent vaccine trials, which have provided insight into the requirements for potential protective immune mechanisms.

GENERAL PRINCIPLES OF AN ANTIVIRAL IMMUNE RESPONSE The degree to which pathogenic organisms establish productive infections is determined in part by the integrity of epithelial and mucosal cells: skin, respiratory tract, alimentary tract, urogenital tract, and conjunctiva. These regions serve as physical barriers between the exterior and internal environment and any abrasions or lesions will allow potential pathogenic organisms into either the blood or lymphatic circulation. Once these physical barriers have been transcended, there are two major categories

45

Section | 1 |

Epidemiology and biology of HIV infection

of host immune responses, namely innate and acquired immunity. The innate immune response represents the first line of defense, and serves to rapidly attenuate the impact of most infectious organisms. From an evolutionary perspective, innate immunity shares properties with lower vertebrate mechanisms of engulfment and phagocytosis and the response consists of specialized cells, such as macrophages, natural killer cells, dendritic cells, and polymorphonuclear leukocytes. Infectious organisms that survive the innate immune response, or residues from such a response, are dealt with by the specific acquired immune response. Acquired immunity has three central tenets: specificity, recognition of protein structures via the interaction of receptors and ligands; diversity, variations in specificity, where multiple receptors interact with different protein structures; and memory, where different T and B cells that have been primed to antigens can be recalled at a subsequent point in time with a more rapid response. What governs specificity and diversity of the adaptive immune response is the genetic make-up of the host, where genes encoding for the major histocompatibility complex (MHC), T cell receptors (TcR), and immunoglobulins (B cell receptors) dictate how and which regions of the pathogen are encountered by the immune system. The molecules encoded by these genes are central to specificity, diversity, and memory and constitute the internal composition of each individual and is collectively known as “self.” An acquired immune response that results in the successful clearance of an invading organism can be understood by the exquisite difference in recognition between “self” and “non-self.” The ultimate outcome of this process is preservation and survival of the species.

ACQUIRED IMMUNITY TO VIRAL INFECTIONS The immune system consists of parallel blood and lymphatic circulations, ensuring that different cells participating in an immune response can migrate back and forth between non-lymphoid tissue and the different secondary lymphoid structures (such as the spleen and lymph nodes). Bone marrow is the primary lymphoid organ, where the precursors to all mature immunocompetent cells are derived as pluripotential progenitor cells. B and T cells develop into mature immunocompetent cells in the bone marrow and thymus, respectively. T cells that leave the thymus are “naı¨ve” and have yet to encounter invading pathogens. Lymph nodes are crucial for providing the correct microenvironment and anatomical structures required for initiating an immune response. The micro-anatomical arrangement of the lymph node enables T cells to encounter processed viral proteins presented by specialized antigen presenting cells, which initiates the adaptive

46

immune response. In general terms, if the anatomical arrangement of lymphoid tissue disintegrates due to pathology, the impact will result in disrupted antigen presentation and loss of both B and T cell priming and the inability to provide protective immunity. Movement of T cells from one lymphoid region to another allows both CD4 and CD8 T cells to encounter processed antigen in the paracortical region of the lymph node. After engaging and processing antigen in the peripheral tissue, dendritic cells will migrate to lymph nodes, where there is selection of reactive T cells through TcR engagement with viral peptides situated in the binding groove of the human leukocyte antigen molecules on the surface of the antigen presenting cells. This process results in multiple clones of expanded T cells, leading to diversity. The MHC in humans is known as the human leukocyte antigen (HLA) system and is one of the most polymorphic proteins in the human population. The uniqueness of individuals is partly defined by HLA, where each person has a defined HLA type consisting of pairs of inherited genes. As the sole function of class I and II HLA is to present processed pathogen-derived peptides, or epitopes, to circulating T cells, possible aberrant T cell function and recognition of self, as in autoimmunity, will thus involve the HLA. HLA class I molecules are co-dominantly expressed on antigen presenting cells, and they play an important role in regulating the fitness of the immune system through a process of selecting and presenting immunogenic peptides to CD8 T cells by TcR recognition (Fig. 4.1A). HLA alleles are X-linked and inherited in pairs (heterozygous), and it is noteworthy that in HIV infection, individuals who are homozygous for one or more alleles (inheriting the same HLA allele from both parents) progress more rapidly to AIDS than heterozygotes [1]. Additionally, HLA-B is more polymorphic than HLA-A and HLA-C and the influence of HLAB is known to have the strongest impact in HIV set point, which is strongly predictive of the rate of progression [2] when compared to HLA-A and HLA-C molecules [3, 4]. The manner by which epitopes are processed and bound by the HLA molecule is highly specific and governed by certain rules associated with the binding motif structures of each HLA and in the correct orientation will be recognized by activated CD8 cytotoxic T lymphocytes (Fig. 4.1A). Sequence changes can occur at anchor positions of targeted epitopes and reduce or interfere with peptide binding to the restricting HLA class I molecule. Moreover, amino acid changes within or immediately adjacent to CD8 T cell epitopes can impede intracellular antigen processing or directly modify the structural interaction between the epitope of HLA class I complex and the TcR of the corresponding CD8 T cells. The ability of viruses to acquire sequence mutations resulting in the loss of recognition by HIV-1-specific CD8 T cells poses a major hurdle for current vaccine efforts. HLA class II molecules have a more restricted distribution and are expressed only on specific cell types and on T cells after activation. Classically, CD4 T cells provide

Chapter

|4|

The immune response to HIV

Activated CD8+ cytotoxic T lymphocyte

TcR Kill

HLA I

Virus

CD8

Tissue cells

Viral protein

"Self" protein

Figure 4.1A Cells infected by viruses or intracellular bacteria are detected and destroyed by CD8 cytotoxic T lymphocytes. CD8 cytotoxic T lymphocytes are activated in the secondary lymphoid organs and migrate to sites of inflammation where they scan cell surfaces with their T cell receptors (TcR) for recognition of foreign peptide antigens displayed on cell surface HLA class I molecules. HLA class I receptors are constitutively expressed on the surface of all cells except erythrocytes. CD8 cytotoxic T lymphocytes destroy target cells by releasing granzymes and perforin and cell-degrading molecules (with permission from Immunopaedia, http://www.immunopaedia.org).

“help” to the immune response by liberating a series of cytokines important for coordinating cellular activity and inducing activated B cells to become antibody-secreting plasma cells (Fig. 4.1B). First identified in murine models, the multitudinous number of cytokines have been organized into a network model of Th1, Th2, Treg, and Th17 cells. A Th1-type response consists of CD4 T cells liberating a profile of cytokines that direct T cell immunity and involves IL-1, IL-2, IL-6, IL-12, IL-15, TNF-a, and IFN-g, for example. A Th-2-type response consists of CD4 T cells liberating a profile of cytokines that directs humoral immune responses and is involved in switching on B cell immunity. These cytokines include, among others, IL-4, IL-5, and IL10. A Th17-type response consists of an IL-17A, IL-17F, and IL-22 profile and is involved in conferring protection against bacteria, fungi, and mycobacteria [5] and to play a role in mucosal defense in the gut. Tregs are CD4þCD25hiFoxP3þ and have been shown to downregulate the activation and proliferation of T cells [5].

A balance exists between pro- and anti-inflammatory immune responses imparted by these CD4 subsets for maintaining the integrity and homeostatic balance of cells in the host. Recently, T helper follicular cells that are involved in the development of antibody-producing plasma cells in the germinal centers of lymphoid tissue have been described [6]. How do these cells fit together in healthy humans? CD8 T cells make up the smaller proportion of CD3 T cells and are involved in protecting the host from invading pathogens. These cells function by killing virally infected cells, which are marked by the surface expression of HLA class I molecules that present virus-derived epitopes that are typically 8-11 amino acids in length (as described above, Fig. 4.1A). These CD8 T cells function with the help of CD4 T helper cells. The killing potential of CD8 T cells is through either perforin/granzyme or Fas–Fas-L interactions and erupted and effete infected cells are engulfed and processed by dendritic cells; virally derived epitopes are presented via cross-presentation [7] by class II HLA

47

Section | 1 |

Epidemiology and biology of HIV infection

Plasma cell

Activated B lymphocyte T cell zone B cell activation signal

4 CD A II HL

Secondary lymphoid organ

R TC Antibodies

CD4 helper T lymphocyte

Figure 4.1B CD4 helper T lymphocytes stimulate B lymphocytes presenting peptide antigens associated with HLA class II receptors in the T cell zone of secondary lymphoid organs. The CD4 helper T lymphocyte provides activation signals to the B lymphocyte to proliferate and differentiate into antibody-secreting plasma cells. Memory B lymphocytes are also generated for long-term immunity (with permission from Immunopaedia, http://www.immunopaedia.org).

molecules and drive CD4 T cell responses. Typically most (99%) expanded viral antigen-specific T cell effector clones induced in the acute phase of infection will die through apoptosis as the immune response wanes, leaving a small residual population of T effector memory cells that migrate to non-lymphoid tissues or T central memory (TCM) cells that recirculate through the lymphatic system and blood circulation and can be rapidly reactivated upon secondary exposure to viral antigens.

IMMUNE RESPONSE TO HIV-1 INFECTION The course of immunological events from the time of transmission can be divided into acute, early, and chronic phases of infection. The greatest challenges HIV presents to the immune system include the selective infection of CD4 T lymphocytes and the extensive viral genetic variability due to mutations. Is HIV a disease of the mucosal immune system? A number of studies have highlighted the importance of the mucosa in HIV pathogenesis and it is now increasingly

48

being recognized as a disease of the mucosal immune system [8], where vaginal and rectal mucosa are the predominant sites of HIV entry and the gut-associated lymphoid tissue (GALT) is the site of initial HIV replication. During the early phase of simian immunodeficiency virus (SIV) and HIV infection, there is a rapid and widespread massive depletion of activated mucosal CD4 T cells at mucosal sites and this occurs before significant depletion in blood and lymph nodes [9]. Recent evidence confirms that the level of CD4 T cell depletion is far higher than at first anticipated, with 60–80% of memory CD4 T cells depleted during early infection. Thus, within the first few weeks of HIV infection, the virus targets the mucosal immune system and dramatically depletes the CD4 T cells at this site. It has also been shown that HIV targeting of activated CD4 T cells in mucosal tissues persists throughout infection, and not just in acute infection as previously thought. Mucosal tissues are likely to be a major source of viral replication, persistence, and continual CD4 T cell loss in HIV-infected individuals. Reduced CD4 T cell frequencies during chronic HIV infection have also been shown in other mucosal sites such as rectal mucosa [10], male genital tract [11], female genital tract [9], and lung mucosa [12].

Chapter

HIV

|4|

The immune response to HIV

Genital epidermaI layer

Genital Iymph node

T cell zone

B cell zone

CD4 helper T lymphocyte

IL-1 IL-6 TNF-α

Squamous epithelial cell layer

Activated epidermal Langerhans cell T cell migration and virus dissemination

Figure 4.2 Epidermal Langerhans cells are a subset of dendritic cells found in the squamous epithelium of the female vagina and male inner foreskin and are the first immune cells to contact HIV during heterosexual contact. They express surface CD207 (langerin) that capture virus by binding to gp120 and induces internalization and degradation of virus. Activated cells migrate to draining lymph nodes for antigen presentation to CD4 T lymphocytes, which can also become infected by surface-bound virus. Langerhans cells also express CD4 and CCR5 and can become infected. Activated Langerhans cells produce pro-inflammatory cytokines IL-1, IL-6, and TNF-a that can cause fever in acute infection (with permission from Immunopaedia, http://www.immunopaedia.org).

Figure 4.2 shows some of the local factors in the mucosal microenvironment that may facilitate HIV replication in mucosal tissues independently from blood: (i) the localized cytokine milieu; (ii) differing inflammatory signals; and (iii) the presence of different immune cell types in these distinct compartments. These factors highlight mucosal sites as critically important in the context of not only understanding HIV pathogenesis but also in terms of being able to possibly dampen or correct the imbalance of proinflammatory signals in potential therapeutic or preventive modalities. During acute HIV infection, there appears to be a hierarchy of systemic immune responses that occur. Figure 4.3 shows a composite schema of the known sequence of immunological responses that occur during infection. After viral transmission (1), where there appears to be a selection of single strain variants at the mucosa [13], there is dissemination (2) of the virus to the lymphoid tissue [14] during the acute phase of infection. Within days after viral transmission, viremia peaks and the downward slope is thought to be a result of a robust cellular immune response leading to initial control (3) of virus [15]. Natural history studies have shown that viral set point is achieved within 6 months of infection and is

prognostic of disease outcome, where high levels of viremia are associated with a more rapid course of infection leading to AIDS. It is noteworthy that seroconversion (4), by the detection of anti-Gag binding antibodies, occurs after peak viremia and that detection of neutralizing antibodies occurs only after approximately 3 months post transmission. In addition to virus-specific CD8 T cells, CD4 T cells appear to be critical for immune control. Animal models of chronic viral infections established that virus-specific CD4 T cells play an essential role in maintenance of effective immunity (reviewed in Day and Walker [16]), and the immune response to HIV appears to follow these same requirements. The detection of enhanced proliferation of anti-HIV-specific CD4 T cells in individuals who maintain long-term control of HIV replication [17] and in patients treated for acute infection with potent antiretroviral therapy [17–19] suggest that the function of these cells is central to influencing viral set point and for controlling virus. As discussed, a large number of CD4 T cells are infected in the gut and that the bulk of the CD4 T cell pool resides within lymphoid tissue around the gastrointestinal tract. Direct killing of CCR5 CD4 T cells within the gut [20, 21] and memory CD4 cells in multiple tissues [22]

49

Section | 1 |

Epidemiology and biology of HIV infection

Chronic infection (asymptomatic)

Acute infection 3 Control

AIDS

CD4 T lymphocytes

Dissemination 2 CD8 T lymphocytes

Neutralizing antibodies

4 Seroconversion

Binding antibodies Transmission

1

Time from infection (months to years)

Figure 4.3 A typical immune response to untreated HIV infection shows a rapid increase in viremia in the acute phase, which declines to a set point. A decline in CD4 T cells coincides with the increase in viral load. HIV-specific CD8 cytotoxic T lymphocyte responses reduce the viral load and an increase in CD4 T cells is observed. HIV-specific binding antibodies appear after the reduction of viremia, but antibodies are detectable by ELISA only later in acute infection. During chronic infection, CD4 T cells decline slowly and viral load remains stable. Neutralizing antibodies begin to appear around 3 months post infection. Continued HIV replication and immune evasion exhausts the immune system, leading to opportunistic infection and AIDS in most infected people, albeit with varying lengths of time (with permission from Immunopaedia, http://www.immunopaedia.org).

by HIV has been postulated as the main mechanism for CD4 T cell depletion during SIV infection in monkeys, and extrapolations to HIV-induced depletion of CD4 T cells within the human gut have also been made [23]. Figure 4.4 shows the potential scenario of microbial translocation from the gut lumen. Studies of HIV/AIDS pathogenesis have long-focused on the role of CD4 T-cell depletion as a key marker of disease progression [24]. The pathogenesis of HIV infection is now characterized by CD4 T cell immunodeficiency in the context of generalized immune activation and dysregulation, with massive memory CD4 T cell infection and depletion during acute infection. This is followed by gradual loss of remaining CD4 T cells caused by persistent immune hyperactivation. Activation of CD4 T cells results in increased target cells for the virus, excessive apoptosis of uninfected T cells, generalized immune dysfunction, and impaired ability to control HIV replication. In addition to cellular immune responses that emerge rapidly after HIV transmission, active humoral immunity is apparent by the increased titers of anti-Gag antibodies, which are used diagnostically to assess seroconversion. Functional antibodies are those that can block or neutralize HIV entry into CD4-bearing cells and such neutralizing antibodies mainly target the highly variable V3 loop, the CD4 binding domain, and the more conserved gp41 transmembrane protein of HIV-1. Although high titers of neutralizing antibodies can completely prevent infection in animal models, the role of these responses in viral containment following infection in humans remains uncertain. During acute infection, these responses appear following the initial drop in viremia, and experimental depletion of B cells in

50

monkeys during acute SIV infection led to delayed emergence of neutralizing antibodies and no change in early viral kinetics [25]. Additionally, passive transfer of neutralizing antibodies in monkeys can protect against intravenous and mucosal challenge of SIV [26]. Thus it is clear that neutralizing antibodies are important for prevention of viral infection, but perhaps less clear once HIV-1 infection has become established. Recent comprehensive longitudinal studies of autologous neutralizing antibody responses following acute infection indicate a significant antiviral effect of these responses, in that the viral inhibitory capacity is of sufficient magnitude to completely replace circulating neutralization-sensitive virus with successive populations of neutralization-resistant virus [33].

WHY THE IMMUNE RESPONSE FAILS TO CONTROL HIV From our discussion so far, HIV infection provokes a burst of immune activity that results in potent targeted cytotoxicT lymphocyte (CTL) responses, CD4 T cell function, and the induction of antibodies. These immunological components have been found to be central role-players in effective clearance of many other viral infections, but fail to clear HIV-1 infection without exception. Why is HIV neither eliminated nor effectively contained following infection? A number of possibilities exist. First, it is important to understand from a virological perspective that HIV-1 establishes persistent infection by infecting CD4-bearing cells

Chapter

|4|

The immune response to HIV

Lamina propria Crypt of Lieberkühn Macrophage Microbial antigens Microbes Translocation

Dendritic cell

Lymphatic vessel

Mucins Antimicrobial defense molecules

Epithelial stem cell

Microvilli

Venule

Paneth cell

Goblet cell

T lymphocyte Arteriole

Enterocyte

Figure 4.4 Activation of the mucosal immune system due to HIV replication in CD4 T helper lymphocytes in the lamina propria leads to the production of pro-inflammatory cytokines. This can cause a disruption of gut epithelial cell development, most notably resulting in villous atrophy and increased gut permeability to microbes and microbial antigens. The consequences of this are malabsorption of nutrients due to reduced absorptive surface area and further stimulation of mucosal immune cells that can enhance HIV replication (with permission from Immunopaedia, http://www.immunopaedia.org).

and integrates into the host genome as provirus. Both primary and secondary lymphoid tissues are targets for seeding by HIV soon after transmission and the anatomical structures within lymph nodes, spleen, and bone marrow are affected by the presence of virus. Consequently, from an immunological perspective, the persistence of antigen directly within the secondary lymphoid structures will influence T and B cell clonality and drive cells into a hyperactivated state. As discussed earlier, the high rate of HIV mutability, due to the error-prone nature of reverse transcriptase, in response to specific B and T cell reactivity, leads to immune escape and contributes to viral persistence within the host. It is also important to understand that almost 30 years after AIDS was first identified, the critical immune functions that control HIV replication remain to be defined. A simplistic view has been that strong CD8 T cell responses, together with strong neutralizing antibodies and virus-specific CD4 T cells, would lead to control of viremia. The breadth and magnitude of CD8 T cell responses do not correlate with control of viremia, at least as measured by IFN-g responses. Although neutralizing antibodies are detectable, there is no simple relationship between these and viremia. Even virusspecific CD4 T cells, which seem critical for maintenance of effective cellular and humoral immune responses in animal models of chronic infection, can be found in some persons with progressive infection. Thus the mechanisms that

account for a lack of long-term control of HIV infection are probably complex. The following are some of the factors for which there is now experimental evidence suggesting that they may participate in the ultimate lack of ability to control HIV replication.

Escape from neutralizing antibodies Despite gradual broadening of the neutralizing antibody response following acute infection, it does not become sufficiently broad to neutralize the next population of virus to arise [27, 28], and the neutralizing antibody responses forever lag behind the evolution of the envelope gene. The relevance of neutralization antibody escape in loss of overall control of viremia is open to debate as escape has also been observed in HIV-infected individuals who persistently control viremia [29, 30].

Escape from CD8 T cell control Studies in acute HIV infection have shown that mutations in the HIV genome occurs rapidly, resulting in viral escape from specific CD8 T cell recognition. During the course of infection the HIV-1 pathogen encodes numerous potentially immunogenic determinants; yet, CD8 T cells only recognize and respond to a minute fraction of potential

51

Section | 1 |

Epidemiology and biology of HIV infection

viral epitopes. These CD8 T cells responses can be classified as immunodominant, co-dominant, and subdominant, depending on their relative contribution to the overall magnitude of HIV-1-specific CD8 T cell responses in a given individual. Data from both human and animal models indicate that during acute infection, highly effective HIV-1-specific CD8 T cells are narrowly directed against selected epitopes and do so in a hierarchal order [31]. This may suggest that the hierarchy of HIV-1-specific CD8 T-cell responses in acute infection may be crucial for the effectiveness of the immune response in chronic infection [31, 32]. Mutations within targeted epitopes [33, 34] can also abrogate established CD8 T cell responses, as can mutations in flanking residues that impair normal antigen processing. Both of these lead to loss of recognition by established T cell responses.

CD8 T cell dysfunction Defects in differentiation and maturation of CTL [35–37] may result in impaired in vivo function, and may relate to lack of CD4 T cells help that is critical for maintenance of effective immunity. Most studies to date have defined CD8 T cell responses based on the ability to secrete IFN-g, which based on animal model data may be the last effector response to be lost by CD8 T cells [38]. While T cells, in particular CD8 T cells, have the ability to carry out many functions in response to antigens including cytotoxicity, release of IFN-g, TNF-a, and MIP-1b, for example, many previous studies on HIV-specific T cell responses focused on measuring one or two functions of these cells. The advancement in flow cytometry recently has allowed the measurement of multiple T cell functions, including degranulation (CD107a mobilization), cytokines (for example IFN-g, IL-2, and TNF-a) and chemokine production (for example, MIP-1b) [39] to determine whether it is the quality of HIV-specific T cell response rather than frequency or breadth that is the important factor in HIV disease progression. Interestingly such studies have found out that non-progression [39] or elite control [40] in HIV infection is associated with maintenance of high levels of polyfunctional CD8 T cells. Other studies further dissected polyfunctionality and demonstrated that it is the upregulation of perforin, a cytolytic enzyme, that is associated with control in HIV infection [41]. Recent data indicate a functional impairment in the ability of CD8 T cells to proliferate to viral antigens in persons with progressive disease that is maintained in persons with non-progressing infection [36], and present in the earliest stages of acute infection when viral load is rapidly declining [42].

Impaired CD4 T cell responses The composition of functionally distinct subsets of virusspecific CD4 T cells, including IL-2- and IL-2/IFN-g-secreting HIV-1-specific CD4 T cells, may be critical [43–45], as may cell killing by CD8 T cells [46]. Infection of a subset

52

of CD8 T cells that are CD4dim following activation has been demonstrated, suggesting that infection of this population of CD8 T cells may contribute to loss of CD8 T cell function in vivo [47, 48].

Impaired dendritic cell function Dendritic cells (DCs) are reported to be among the first cells with which HIV-1 interacts and through expression of a diverse array of receptors and signaling molecules, DC’s capture and process HIV-1, and present associated antigens to T cells. DCs serve as crucial links between innate and adaptive immune responses through expression of pattern-recognition receptors (PRRs) such as Toll-like receptors (TLRs) and intracellular pathogen sensors, which trigger specific signaling pathways that lead to host defense. There are also recent studies in the SIV model and in a small human clinical trial suggesting impaired induction of immune responses in chronic infection, which may be related to the impaired function of DC during viral infection. These studies [49], in which adoptive transfer of in vitro matured DCs loaded with inactivated virus led to a decrease in set point viremia and increase in virus-specific immune responses, need to be confirmed by additional groups, but suggest that there is impairment in the inductive phase of the immune response.

Lymphoid structure degeneration After infection, HIV-1 resides within the germinal centers of the lymph nodes, spleen, tonsils, and thymus and exists, in the main, as whole virus on the surface of follicular dendritic cells (FDCs) in the lymph node. As a result, the normal function of FDC in presenting antigen may be overridden. Viral transmission is thought to be cell-associated and productive infection most likely takes place in lymphoid structures in close proximity to the cervix and rectum, where DCs from the newly infected individual migrate from the cervical region to lymph nodes elsewhere in the body. Complete virus is most likely carried on DCs by dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), which aids in transmission to T cells [50] in the lymph nodes distal to the point of viral transmission. Although there are no clear data relating the dissolution of the lymph node architecture with persistence of virus, and ongoing immune responses, it may be hypothesized that destruction of the platform used to initiate immune responses leads to an overall failure in T cell priming to new antigens. Evidence from regenerated lymphoid structures after successful antiretroviral treatment supports this notion [51].

Other concomitant infections The extent to which the ability to control HIV may be impaired by other concomitant infections is a particularly relevant question for those areas where the epidemic is

Chapter

|4|

The immune response to HIV

currently most rapidly expanding. In many regions of the world, co-infections with Mycobacterium tuberculosis (mTB) [52] and other co-viral infections are predominant, and the impact of co-infections on host immunity to HIV is also relatively unknown. Herpes simplex virus type 2 (HSV-2) infection, for example, results in a persistent localized inflammatory response in the dermis below the healed lesion, consisting of HSV-2-specific CD4 T cells that express CCR5 or CXCR4, which are important HIV co-receptors. [53] Furthermore, HSV-2 suppressive therapy was shown to significantly reduce plasma viral loads and reduce genital tract HIV shedding in HIV-infected women co-infected with HSV-2 [54]. Human papilloma virus (HPV) infection of the cervix may influence HIV pathogenesis by inducing the production of immune and inflammatory factors that enhance HIV expression [55]. A meta-analysis was recently performed on 39 different studies that reported the effect of genital tract infections on the detection of HIV shedding in the genital tract [56]. In this meta-analysis, HIV1 detection in the genital tract was increased most substantially by urethritis and cervicitis. Cervical discharge or mucopus, gonorrhoea, chlamydial infection, and vulvovaginal candidiasis were also significantly associated with HIV shedding. Alternatively, some studies have shown that there may be beneficial protective effects of HIV co-infection with GB virus C (GBV-C) [57], where protection may result from the induction of different chemokines that have anti-HIV properties. However, the mortality rates within cohort studies in populations of mine workers in southern Africa who are co-infected with mTB and HIV suggest that co-bacterial infections are anything but protective [58, 59].

EVIDENCE FOR CORRELATES OF PROTECTION TO HIV-1 INFECTION Even though HIV-1 infection establishes persistence and undermines immunity, there are a small number of people who are infected but are clinically healthy and can control viremia to extraordinarily low levels: to less than 50 copies/ mL plasma. Additionally, there are also individuals who have frequent high-risk sexual encounters and who have a high probability of being exposed to HIV, but remain uninfected. These two cohorts of individuals may provide clues to which factors of host immunity correlate with delayed disease or possible protection from infection [57]. Identity of these immune factors is important for providing clues to protective immunity and for devising successful vaccine strategies. Several cohort studies, including those of highly exposed persistently seronegative (HEPS) sex workers, occupationally exposed healthcare workers, and uninfected babies born to HIV-infected mothers, have reported evidence for systemic and mucosal

cytotoxic T cell activity and chemokine receptor mutations as significant markers associating with their seronegative status. Less well-associated factors such as chemokine production, HLA alleles/haplotypes, helper T cell responses, humoral responses, and soluble inhibitory factors have been described, but less uniformly. Evidence for mucosal HIV-specific IgA antibodies in HEPS individuals also provide evidence for possible protective roles of local antibody responses—although these studies often have examined small numbers of individuals [60]. A more recent exploration of defining which immune factors correlate with protection, or attenuation of disease in HIV-infected individuals, has been to identify human genes with polymorphic variants that influence the outcome of HIV-1 exposure or infection. Several AIDSrestricting genes (ARG) have been identified within the human genome that are related to either resistance or acceleration of disease (reviewed by O’Brien and Nelson [61]). Unbiased assessment of genes involved in viral control in HIV-infected people, using a whole genome association strategy, have strongly associated specific HLA genes, most notably HLA-B*57-01, with viral set point.

PROSPECTS FOR VACCINES A vaccine is of paramount importance to develop and implement as a public health option in many regions of the globe where HIV-1 incidence rates are extremely high. If a preventive vaccine can lower the rate of secondary viral transmission, this will have a significant effect of mitigating the epidemic. Although successful vaccines have been implemented for diseases such as smallpox, polio, measles, and hepatitis B, replicating these designs for HIV-1 may not be appropriate. Persistence of HIV leading to chronic infection and the continuous viral evolution in the infected host, to evade antibody and CTL responses, needs to be accounted for. It is known, for example, that chronic persistence of antigen leads to diminished central memory T cells, and the inability of the immune system to mount an effective secondary response [62]. Thus a vaccine would be required to enhance the pool of central memory, either in a preventive nature or as a therapeutic option. Development of therapeutic vaccines is extremely relevant for the large numbers of HIV-1-infected individuals, where the aim would be to redirect immunity under the cover of antiretroviral therapy to effectively contain infection. There is no doubt that a successful preventive vaccine will need to evoke aspects of innate, cellular, and humoral immunity. As the major determining factor of disease progression in an individual is viremia and the level of infectiousness is proportional to the magnitude of the viral load in plasma, genital tract secretions, and breast-milk, a vaccine capable of controlling viral replication would

53

Section | 1 |

Epidemiology and biology of HIV infection

potentially lower the rate of disease progression and secondary transmission. Typically a phase IIb test-of-concept or III efficacy vaccine trial measures the candidate vaccine relative to a placebo control arm. The primary end-point measurement is lower incidence in the vaccine arm, although it is more likely that a secondary end-point will be levels of viremia at a specific time post infection and the time to set point. The recently halted STEP study, which was a double-blind phase II test-of-concept trial in 3,000 HIV seronegative volunteers in North America, the Caribbean, South America, and Australia receiving three injections of an adenovirus vector (MRKAd5) containing subtype B gag/pol/nef genes, failed to show efficacy and made no impact on levels of viremia at 3 months after vaccination in those who became infected [63]. Early followup showed that the vaccine arm may have been deleterious and hinted at promoting infection, but over longer-term follow-up, the number of infections per study arm became non-significant. The one factor associated with infection was whether men were circumcised [63], with the uncircumcised male being more susceptible. Whether this has to do with epidermal Langerhans cells in the foreskin that can trap virus (Fig. 4.2) is open to question. A companion trial in South Africa, the Phambili trial, showed a HIV infectivity pattern similar to that of the STEP study, with the major difference being that more women than men were enrolled and thus the enhanced infectivity in the vaccine arm in this trial was not related to circumcision status [64]. In contrast to the failure of the STEP and Phambili studies to show vaccine efficacy was the modest success of the prime boost phase III RV144 trial in Thailand [65]. This trial consisted of priming with a recombinant canarypox vector vaccine (ALVAC-HIV vCP1521) plus two booster doses of a recombinant glycoprotein 120 subunit (AIDSVAX B/E) in 16,402 HIV-1 seronegative men and women in Thailand. The recombinant gp120 immunogen alone showed no efficacy in a large earlier trial in Thailand [66], but when combined with the ALVAC product as a boost, showed a 31.2% efficacy using a modified intentto-treat analysis [65]. Through the combined analyses of the STEP/Phambili and RV144 trial results, there will hopefully be insight into immune responses that are, and are not, important for vaccine efficacy. However, what clues do we have so far of the kind of immune response that will be required from a vaccine? The immune responses detected in monkeys as well as those detected in HEPS individuals and long-term non-progressors suggest that an effective HIV vaccine will need to elicit neutralizing antibodies, CD4 T cell responses, and CTL [52]. As discussed, since the major route of viral transmission is through mucosal barriers, and that HIV may be regarded as a disease of mucosal immunity, it will be crucial to elicit mucosal vaccine-induced immunity and not only systemic immunity. One feature of a vaccine-induced response is the elicitation of long-lived memory CD4 and CD8 T cells that can be rapidly recalled

54

in the event of a subsequent exposure or infection with HIV-1. The aim of an HIV vaccine response would be to generate pools of vaccine-specific long-lived central memory CD8 T cells. As discussed above, the persistence of antigen upon HIV infection compromises the development of central memory CD8 T cells, whereas the transient nature of vaccine-related antigens would create a more favorable environment for induction of central memory. Other vaccine candidates are either in early clinical trials or in the pipeline of development (http://www.iavi.org, http://www.hvtn.org, http://www.chavi.org, and http:// www.cavd.org). Whatever the eventual mechanisms leading to successful vaccine immunity, mucosal responses that can neutralize or contain HIV at the point of entry will need to be elicited, to cater for the genetic heterogeneity of HIV-1 across the globe, and to generate long-term memory. Notwithstanding these challenging scientific issues, uniform access of a stable vaccine to all individuals at an affordable cost is a social and political challenge that needs to be addressed in tandem with scientific development.

CONCLUSION HIV infection elicits robust cellular and humoral immune responses, but the vast majority of persons ultimately fail to control viremia and progress to AIDS. Although progress has been made in understanding the reasons for ultimate lack of control, there are critical gaps in our knowledge that need to be resolved to facilitate rational vaccine design, and to guide immunotherapeutic interventions. Among these are the critical ratios of humoral and cellular immune responses, the critical viral antigens to target, the means to induce broadly cross-reactive protective immunity to the multiple strains currently fueling the global epidemics, and vaccine vector systems that are able to elicit potent antiviral immune responses. Great strides have been made recently in the detection, isolation, and crystallization of broadly neutralizing antibodies [67–70] and the design of candidate immunogens based on the structures of these antibody molecules. With a modestly successful vaccine strategy and learning from trial failures, as well as major scientific advances in the structure and function of broadly neutralizing antibodies, there is reason for optimism. Emerging data also suggest that HIV may not be infinitely mutable, but that there are predictable mutations that occur within given residues when they come under immune selection pressure. Whether this knowledge can lead to the refinement of a vaccine strategy that would provide sufficient protection against circulating viruses remains to be determined. In the meantime, HIV will remain the most significant infectious disease of our generation, and will continue to extract a disproportionate toll on those most marginalized and disenfranchised in each society.

Chapter

|4|

The immune response to HIV

REFERENCES [1] Carrington M, Nelson GW, Martin MP, et al. HLA and HIV-1: heterozygote advantage and B*35Cw*04 disadvantage. Science 1999;283:1748–52. [2] Mellors JW, Rinaldo Jr. CR, Gupta P, et al. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 1996;272:1167–70. [3] Goulder PJ, Watkins DI. Impact of MHC class I diversity on immune control of immunodeficiency virus replication. Nature Rev Immunol 2008;8:619–30. [4] Kiepiela P, Leslie AJ, Honeyborne I, et al. Dominant influence of HLA-B in mediating the potential coevolution of HIV and HLA. Nature 2004;432:769–75. [5] Prendergast A, Prado JG, Kang YH, et al. HIV-1 infection is characterized by profound depletion of CD161þ Th17 cells and gradual decline in regulatory T cells. AIDS 2010;24:491–502. [6] Batten M, Ramamoorthi N, Kljavin NM, et al. IL-27 supports germinal center function by enhancing IL-21 production and the function of T follicular helper cells. J Exp Med 2010;207(13):2895–906. [7] Melief CJ. Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? Eur J Immunol 2003;33:2645–54. [8] Derdeyn CA, Silvestri G. Viral and host factors in the pathogenesis of HIV infection. Curr Opin Immunol 2005;17:366–73. [9] Veazey RS, Marx PA, Lackner AA. Vaginal CD4þ T cells express high levels of CCR5 and are rapidly depleted in simian immunodeficiency virus infection. J Infect Dis 2003;187:769–76. [10] Critchfield JW, Young DH, Hayes TL, et al. Magnitude and complexity of rectal mucosa HIV-1specific CD8þ T-cell responses during chronic infection reflect clinical status. PLoS One 2008;3: e3577. [11] Politch JA, Mayer KH, Anderson DJ. Depletion of CD4þ T cells in semen

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

during HIV infection and their restoration following antiretroviral therapy. J Acquir Immune Defic Syndr 2009;50:283–9. Vajdy M, Veazey R, Tham I, et al. Early immunologic events in mucosal and systemic lymphoid tissues after intrarectal inoculation with simian immunodeficiency virus. J Infect Dis 2001;184:1007–14. Keele BF, Giorgi EE, SalazarGonzalez JF, et al. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 2008;105:7552–7. Gray CM, Mlotshwa M, Riou C, et al. Human immunodeficiency virusspecific gamma interferon enzymelinked immunospot assay responses targeting specific regions of the proteome during primary subtype c infection are poor predictors of the course of viremia and set point. J Virol 2009;83:470–8. Kemal KS, Burger H, Mayers D, et al. HIV-1 drug resistance in variants from the female genital tract and plasma. J Infect Dis 2007;195:535–45. Day CL, Walker BD. Progress in defining CD4 helper cell responses in chronic viral infections. J Exp Med 2003;198:1773–7. Rosenberg ES, Billingsley JM, Caliendo AM, et al. Vigorous HIV-1specific CD4þ T cell responses associated with control of viremia. Science 1997;278:1447–50. Malhotra U, Berrey MM, Huang Y, et al. Effect of combination antiretroviral therapy on T-cell immunity in acute human immunodeficiency virus type 1 infection. J Infect Dis 2000;181:121–31. Oxenius A, Price DA, Easterbrook PJ, et al. Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8þ and CD4þ T lymphocytes. Proc Natl Acad Sci U S A 2000;97:3382–7.

[20] Veazey RS, Lackner AA. HIV swiftly guts the immune system. Nat Med 2005;11:469–70. [21] Li Q, Duan L, Estes JD, et al. Peak SIV replication in resting memory CD4þ T cells depletes gut lamina propria CD4þ T cells. Nature 2005;434:1148–52. [22] Mattapallil JJ, Douek DC, Hill B, et al. Massive infection and loss of memory CD4þ T cells in multiple tissues during acute SIV infection. Nature 2005;434:1093–7. [23] Mehandru S, Poles MA, TennerRacz K, et al. Primary HIV-1 infection is associated with preferential depletion of CD4þ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 2004;200:761–70. [24] Sodora DL, Silvestri G. HIV, mucosal tissues, and T helper 17 cells: where we come from, where we are, and where we go from here. Curr Opin HIV AIDS 2010;5:111–13. [25] Schmitz JE, Kuroda MJ, Santra S, et al. Effect of humoral immune responses on controlling viremia during primary infection of rhesus monkeys with simian immunodeficiency virus. J Virol 2003;77:2165–73. [26] Mascola JR, Stiegler G, VanCott TC, et al. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 2000;6:207–10. [27] Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci U S A 2003;100:4144–9. [28] Wei X, Decker JM, Wang S, et al. Antibody neutralization and escape by HIV-1. Nature 2003;422:307–12. [29] Bradney AP, Scheer S, Crawford JM, et al. Neutralization escape in human immunodeficiency virus type 1-infected long-term nonprogressors. J Infect Dis 1999;179:1264–7. [30] Montefiori DC, Altfeld M, Lee PK, et al. Viremia control despite escape from a rapid and potent autologous

55

Section | 1 |

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

56

Epidemiology and biology of HIV infection

neutralizing antibody response after therapy cessation in an HIV-1infected individual. J Immunol 2003;170:3906–14. Streeck H, Jolin JS, Qi Y, et al. Human immunodeficiency virus type 1-specific CD8þ T-cell responses during primary infection are major determinants of the viral set point and loss of CD4þ T cells. J Virol 2009;83:7641–8. Allen TM, O’Connor DH, Jing P, et al. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature 2000;407:386–90. Yokomaku Y, Miura H, Tomiyama H, et al. Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection. J Virol 2004;78:1324–32. Kimura Y, Gushima T, Rawale S, et al. Escape mutations alter proteasome processing of major histocompatibility complex class Irestricted epitopes in persistent hepatitis C virus infection. J Virol 2005;79:4870–6. Champagne P, Ogg GS, King AS, et al. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 2001;410:106–11. Migueles SA, Laborico AC, Shupert WL, et al. HIV-specific CD8þ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol 2002;3:1061–8. Appay V, Dunbar PR, Callan M, et al. Memory CD8þ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med 2002;8:379–85. Wherry EJ, Blattman JN, MuraliKrishna K, et al. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol 2003;77:4911–27. Betts MR, Nason MC, West SM, et al. HIV nonprogressors preferentially maintain highly functional HIVspecific CD8þ T cells. Blood 2006;107:4781–9.

[40] Owen RE, Heitman JW, Hirschkorn DF, et al. HIVþ elite controllers have low HIV-specific Tcell activation yet maintain strong, polyfunctional T-cell responses. AIDS 2010;24:1095–105. [41] Hersperger AR, Pereyra F, Nason M, et al. Perforin expression directly ex vivo by HIV-specific CD8þ T-cells is a correlate of HIV elite control. PLoS Pathog 2010;6:e1000917. [42] Lichterfeld M, Kaufmann DE, Yu XG, et al. Loss of HIV-1-specific CD8þ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4þ T cells. J Exp Med 2004;200:701–12. [43] Boaz MJ, Waters A, Murad S, et al. Presence of HIV-1 Gag-specific IFNgammaþIL-2þ and CD28þIL-2þ CD4 T cell responses is associated with nonprogression in HIV-1 infection. J Immunol 2002;169:676–85. [44] Iyasere C, Tilton JC, Johnson AJ, et al. Diminished proliferation of human immunodeficiency virusspecific CD4þ T cells is associated with diminished interleukin-2 (IL2) production and is recovered by exogenous IL-2. J Virol 2003;77:10900–9. [45] Harari A, Petitpierre S, Vallelian F, Pantaleo G. Skewed representation of functionally distinct populations of virus-specific CD4 T cells in HIV1-infected subjects with progressive disease: changes after antiretroviral therapy. Blood 2004;103:966–72. [46] Boritz E, Palmer BE, Wilson CC. Human immunodeficiency virus type 1 (HIV-1)-specific CD4þ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels. J Virol 2004;78:12638–46. [47] Cochrane A, Imlach S, Leen C, et al. High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo. J Virol 2004;78:9862–71. [48] Zloza A, Sullivan YB, Connick E, et al. CD8þ T cells that express CD4 on their surface (CD4dimCD8bright T cells) recognize an antigen-specific target, are detected in vivo, and can be

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

productively infected by T-tropic HIV. Blood 2003;102:2156–64. Lu W, Wu X, Lu Y, et al. Therapeutic dendritic-cell vaccine for simian AIDS. Nat Med 2003;9:27–32. Arrighi JF, Pion M, Garcia E, et al. DC-SIGN-mediated infectious synapse formation enhances X4 HIV-1 transmission from dendritic cells to T cells. J Exp Med 2004;200:1279–88. Gray CM, Lawrence J, Ranheim EA, et al. Highly active antiretroviral therapy results in HIV type 1 suppression in lymph nodes, increased pools of naive T cells, decreased pools of activated T cells, and diminished frequencies of peripheral activated HIV type 1-specific CD8þ T cells. AIDS Res Hum Retroviruses 2000;16:1357–69. Kaufmann SH, McMichael AJ. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat Med 2005;11: S33–S44. Zhu J, Hladik F, Woodward A, et al. Persistence of HIV-1 receptorpositive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med 2009;15:886–92. Anderson BL, Cu-Uvin S. Determinants of HIV shedding in the lower genital tract of women. Curr Infect Dis Rep 2008;10:505–11. Smith JS, Moses S, Hudgens MG, et al. Increased risk of HIV acquisition among Kenyan men with human papillomavirus infection. J Infect Dis 2010;201:1677–85. Johnson LF, Lewis DA. The effect of genital tract infections on HIV-1 shedding in the genital tract: a systematic review and metaanalysis. Sex Transm Dis 2008;35:946–59. Kulkarni PS, Butera ST, Duerr AC. Resistance to HIV-1 infection: lessons learned from studies of highly exposed persistently seronegative (HEPS) individuals. AIDS Rev 2003;5:87–103. Day JH, Grant AD, Fielding KL, et al. Does tuberculosis increase HIV load? J Infect Dis 2004; 190:1677–84.

Chapter

|4|

The immune response to HIV

[59] Corbett EL, Charalambous S, Moloi VM, et al. Human immunodeficiency virus and the prevalence of undiagnosed tuberculosis in African gold miners. Am J Respir Crit Care Med 2004;170:673–9. [60] Kaul R, Plummer F, Clerici M, et al. Mucosal IgA in exposed, uninfected subjects: evidence for a role in protection against HIV infection. AIDS 2001;15:431–2. [61] O’Brien SJ, Nelson GW. Human genes that limit AIDS. Nat Genet 2004;36:565–74. [62] Garber DA, Silvestri G, Feinberg MB. Prospects for an AIDS vaccine: three big questions, no easy answers. Lancet Infect Dis 2004;4:397–413. [63] Buchbinder SP, Mehrotra DV, Duerr A, et al. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a doubleblind, randomised, placebo-

controlled, test-of-concept trial. Lancet 2008;372:1881–93. [64] Gray G, Buchbinder S, Duerr A. Overview of STEP and Phambili trial results: two phase IIb test-ofconcept studies investigating the efficacy of MRK adenovirus type 5 gag/pol/nef subtype B HIV vaccine. Curr Opin HIV AIDS 2010;5:357–61. [65] Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009;361:2209–20. [66] Pitisuttithum P, Gilbert P, Gurwith M, et al. Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis 2006;194:1661–71.

[67] Pancera M, McLellan JS, Wu X, et al. Crystal structure of PG16 and chimeric dissection with somatically related PG9: structure–function analysis of two quaternary-specific antibodies that effectively neutralize HIV-1. J Virol 2010;84:8098–110. [68] Wu X, Yang ZY, Li Y, et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 2010;329:856–61. [69] Zhou T, Georgiev I, Wu X, et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 2010;329:811–17. [70] Walker LM, Phogat SK, ChanHui PY, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 2009;326 (5950):285–9.

57