Improved protection conferred by vaccination with a recombinant vaccinia virus that incorporates a foreign antigen into the extracellular enveloped virion

Virology 322 (2004) 337 – 348 www.elsevier.com/locate/yviro

Improved protection conferred by vaccination with a recombinant vaccinia virus that incorporates a foreign antigen into the extracellular enveloped virion Heesun Kwak, a,1,2 Waleed Mustafa, b,1 Kendra Speirs, a,1 Asha J. Abdool, a Yvonne Paterson, b and Stuart N. Isaacs a,* a

Division of Infectious Diseases, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6073, USA b Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA Received 1 August 2003; returned to author for revision 18 August 2003; accepted 16 February 2004

Abstract Recombinant poxviruses have shown promise as vaccine vectors. We hypothesized that improved cellular immune responses could be developed to a foreign antigen by incorporating it as part of the extracellular enveloped virion (EEV). We therefore constructed a recombinant vaccinia virus that replaced the cytoplasmic domain of the B5R protein with a test antigen, HIV-1 Gag. Mice immunized with the virus expressing Gag fused to B5R had significantly better primary CD4 T-cell responses than recombinant virus expressing HIV-Gag from the TK-locus. The CD8 T-cell responses were less different between the two groups. Importantly, although we saw differences in the immune response to the test antigen, the vaccinia virus-specific immune responses were similar with both constructs. When groups of vaccinated mice were challenged 30 days later with a recombinant Listeria monocytogenes that expresses HIV-Gag, mice inoculated with the virus that expresses the B5R-Gag fusion protein had lower colony counts of Listeria in the liver and spleen than mice vaccinated with the standard recombinant. Thus, vaccinia virus expressing foreign antigen incorporated into EEV may be a better vaccine strategy than standard recombinant vaccinia virus. D 2004 Elsevier Inc. All rights reserved. Keywords: Vaccinia virus; Recombinant vaccine; EEV; B5R chimeric protein; HIV; Gag; Cellular immune response; Listeria monocytogenes; Heterologous challenge

Introduction Recombinant poxviruses have shown promise as effective vaccine vectors because of their ability to prime adaptive immune responses to expressed foreign antigens. Enhancement of the immune response to the foreign antigen has been pursued as a way to further improve vaccine efficacy. Approaches to augment the immune response have ranged from the co-expression of cytokines or other immune modulating molecules along with the foreign antigen (Car* Corresponding author. Division of Infectious Diseases, Department of Medicine, University of Pennsylvania School of Medicine, 502 Johnson Pavilion, Philadelphia, PA 19104-6073. Fax: +1-215-349-5111. E-mail address: [email protected] (S.N. Isaacs). 1 These authors contributed equally to this study. 2 Current address: Department of Surgery, Columbia University College of Physicians and Surgeons, 177 Fort Washington Avenue, New York, NY 10032. 0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2004.02.007

roll et al., 1998; Honda et al., 1998; Kaufman et al., 2002; McLaughlin et al., 1997; Rodriguez et al., 1999; Sivanandham et al., 1998) to prime-boost strategies (e.g., (Amara et al., 2001; Oh et al., 2003)). In recent years, there has been interest in incorporating the foreign antigen as part of the vaccinia virus particle (Barchichat and Katz, 2002; Katz et al., 1997). The majority of poxvirus-based vaccines produce antigens that remain in the cytoplasm, are secreted, or are expressed on the outer membrane of infected cells. Enhancing the immune response through expression of foreign antigens on particles or virions has been shown in other systems (Allsopp et al., 1996; Beneduce et al., 2002; Casal, 1999; Evans et al., 1989; Mandl et al., 1998; Ulrich et al., 1998). For example, particles of hepatitis B virus when used to carry foreign epitopes induced protective B and T-cell immune responses against the inserted antigens (Ulrich et al., 1998). For poxviruses, a single study

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showed that a recombinant vaccinia virus displaying the HIV-1 envelope glycoprotein on the surface of the extracellular enveloped virus (EEV) induced higher HIV-specific antibody responses in mice than a standard vaccinia virus vector expressing an unmodified HIV glycoprotein that is not trafficked to EEV (Katz and Moss, 1997). Cellular immune responses to this antigen have not been investigated. The B5R protein is one of a group of EEV-specific proteins encoded by vaccinia virus (Engelstad and Smith, 1993; Engelstad et al., 1992; Isaacs et al., 1992; MartinezPomares et al., 1993; Smith et al., 2002; TakahashiNishimaki et al., 1991; Wolffe et al., 1993). Specific domains of B5R are dispensable for EEV formation, allowing for the generation of B5R chimeras that target proteins to EEV (Herrera et al., 1998; Katz et al., 1997; Lorenzo et al., 1998; Mathew et al., 1998, 2001; Rodger and Smith, 2002). We and others have shown that EEV is still formed in the absence of the cytoplasmic tail of the B5R protein (Lorenzo et al., 1998; Mathew et al., 1999, 2001). For this study, we substituted the cytoplasmic domain of the B5R protein with a foreign protein, HIV-1 Gag, and found that Gag was specifically incorporated into EEV. T-cell responses to the B5R-Gag chimera were enhanced and conferred better protection from a heterologous challenge compared to a standard recombinant vaccinia virus that does not incorporate Gag into EEV. Thus, this report is the first to illustrate that cellular immunity to a foreign antigen is improved when that antigen is expressed as part of EEV.

Results Expression of B5R-Gag fusion protein results in incorporation of Gag into EEV We successfully constructed and isolated a recombinant vaccinia virus (strain IHDJ) that replaced the cytoplasmic tail of the B5R protein with the HIV-Gag sequence. The resulting virus (rVV-B5R/Gag) generated smaller plaques under semisolid overlay. Growth curves at low and high multiplicity of infection (m.o.i.) showed that when compared to wild-type virus (WT) or the control virus that expresses HIV-Gag from the TK-locus (rVV-TK/Gag), rVVB5R/Gag released approximately 10 times less virus in the media (Figs. 1A and C). Growth curves of cell-associated virus by wild type, rVV-TK/Gag, and rVV-B5R/Gag were similar (Figs. 1B and D). Thus, despite the decreased amount of released virus by rVV-B5R/Gag, at low m.o.i., enough virus is being released to efficiently spread the virus in tissue culture. Western blotting with anti-B5R antibody of infected cell lysate from the recombinant virus producing a chimeric B5R/Gag protein showed a fusion protein of the predicted molecular weight (approximately 85 kDa) (data not shown). When the blot was stripped and probed with a rabbit polyclonal antiserum to Gag, the same 85-kDa band was seen (data not shown). To determine the amount of Gag antigen synthesized by the rVV-B5R/Gag and rVV-TK/Gag, we quantitated Gag/p24 levels in infected cell lysates. rVVTK/Gag produced moderately higher (4– 6) levels of Gag than rVV-B5R/Gag (data not shown). This is likely due to

Fig. 1. Growth curves of viruses at low and high multiplicities of infection. Confluent BSC-1 cells were infected at an m.o.i. of 0.005 (A and B) or 5 (C and D). At the indicated time points, virus contained in the media (A and C) or cell lysate (B and D) were titered. Open squares: rVV-B5R/Gag; open circles with dashed line: rVV-TK/Gag; closed triangle: wild-type virus.

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the differences in the promoters used to drive expression of Gag. Cesium chloride gradients of media from metabolically labeled cells infected with the viruses revealed that rVVB5R/Gag produced EEV at levels about 5-fold lower than wild type (Fig. 2). Western blotting of EEV released into the media of infected cells with an anti-Gag antibody revealed that rVV-B5R/Gag incorporated Gag into the virion, although wild type and rVV-TK/Gag did not (Fig. 3). Vaccinia virus-specific immune responses to rVV-B5R/Gag and rVV-TK/Gag are identical rVV-B5R/Gag and rVV-TK/Gag are genetically distinct recombinant viruses. Vaccinia viruses with an inactivated TK gene are attenuated (Buller et al., 1985). Because the standard recombinant vaccinia virus expressing Gag has an inactivated TK gene and the recombinant vaccinia virus expressing B5R/Gag has an intact TK gene, we examined the vaccinia virus-specific immune responses of each recombinant virus between ensure that differences we found to the Gag-specific immune responses (see below) did not result from an improved immune response to all antigens in one vaccine vs. the other. Cell-mediated immunity, as measured by IFN-g production, antigen-specific proliferation, and cytotoxic T lymphocyte (CTL)-mediated target cell

Fig. 2. Quantitation of EEV released from infected cells by cesium chloride gradient centrifugation. Confluent RK-13 cells were infected with (A) rVVB5R/Gag or (B) wild type (IHDJ) virus in the presence of S-35 cysteine and methionine. Twenty-four hours postinfection, media was placed over a CsCl gradient and spun at approximately 111,000  g for 3 h. Fractions were collected from the bottom of the tube and the radioactivity was counted in a scintillation counter (closed squares). The density (open circles) of alternating fractions was determined to confirm that the peak activity was similar.

Fig. 3. Western blot of EEV. The proteins contained in EEV from the media of cells infected with wild-type virus (WT) or the recombinant virus expressing Gag fused to B5R (B5R/Gag) or Gag expressed in the TK region (TK/Gag) were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Gag antibody. Arrow to the right indicates the B5R-Gag fusion protein. Numbers on the left refer to the molecular masses (in kilodaltons). To confirm that approximate equal amounts of EEV were loaded in each lane, the blot was stripped and probed with an anti-P37 antibody (lower blot).

lysis, was equivalent between rVV-B5R/Gag and rVV-TK/ Gag immunized mice (Figs. 4A – C). The antibody responses mounted against each recombinant virus were also comparable (Fig. 4D). It should be noted that the cellmediated and humoral immune responses elicited in response to both recombinant viruses were significantly reduced from those mounted against WT virus (data not shown). Thus, rVV-B5R/Gag and rVV-TK/Gag generate equivalent vector-specific immune responses. To determine whether the weakened virulence of the recombinant viruses impaired their ability to confer protective immunity to vaccinia virus challenge, we immunized mice with rVVB5R/Gag or rVV-TK/Gag. After 1 month, we challenged the mice with a lethal dose of vaccinia and measured weight loss over time. Although unimmunized mice succumbed to disease between 4 and 6 days postinfection, vaccinated animals were rendered equally resistant (Fig. 4E). Gag-specific CD4+ T-cell proliferative responses is improved in mice immunized with rVV-B5R/Gag To measure Gag-specific CD4+ T-cell proliferation, we incubated splenocytes from rVV-B5R/Gag, rVV-TK/Gag, or

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Fig. 4. Vaccinia-specific immune responses. Splenocytes from immunized mice were harvested 2 weeks after vaccination and co-cultured with naı¨ve antigenpresenting cells (APC) that had been infected with WT virus (INF APC) or left uninfected (unINF APCs). (A) IFN-g ELISpot. (B) Antigen-specific proliferation measured by BrdU incorporation. (C) CTL responses were determined by restimulating splenocytes with vaccinia for 6 days. Cells were harvested, labeled with Calcein AM, and co-cultured with infected P815 targets at 50:1, 25:1, 12.5:1 (splenocyte/target) ratio. After 3 h, Calcein release was quantitated using a fluorometer and percentage of specific lysis determined. (D) Anti-vaccinia virus antibody responses in sera from mice immunized 2 weeks earlier were measured by ELISA. Absorbance values of serum samples correspond to a 1/30 dilution. (E) Immunized mice are protected from a vaccinia challenge. BALB/c mice were unimmunized or immunized intraperitoneally with rVV-B5R/Gag, rVV-TK/Gag, or WT virus. Mice were challenged intranasally with 2  106 pfu of WT virus (strain WR) and the percentage of weight loss calculated over time. Shown are the means F SEM of individual mice for each condition. Each experiment was performed twice with similar results.

WT immunized mice with a class II immunodominant Gag peptide (Mata and Paterson, 1999) and quantitated proliferation by a colorimetric assay. Pigeon cytochrome C peptide (a class II peptide) and con-A were used as controls. Splenocytes from all three immunized groups responded well to con-A, with a specific index (S.I.) well above 15, but did not respond to irrelevant control peptide (data not

shown). However, splenocytes from mice immunized with rVV-B5R/Gag showed significantly improved proliferation in response to class II Gag-specific peptide than those from mice immunized with rVV-TK/Gag ( P < 0.0001) (Fig 5). Thus, immunization with a virus in which the foreign antigen was incorporated into the virion elicited a statistically better CD4+ T-cell proliferation response.

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Fig. 5. Gag-specific CD4+ T-cell proliferative responses from immunized mice. Ten days after immunization, splenocytes of mice immunized with rVV-B5R/ Gag or rVV-TK/Gag or wild-type control virus were incubated with class II Gag peptide for 48 h. Stimulation Index (S.I.) was calculated as O.D. with antigen/ O.D. without antigen. Five mice were in each group per experiment and result shown is the representative from three separate experiments. Error bars represent standard deviation (rVV-B5R/Gag vs. rVV-TK/Gag, P < 0.0001; rVV-B5R/Gag vs. wild type, P < 0.0001; rVV-TK/Gag vs. wild type, P = 0.1813).

rVV-B5R/Gag immunization results in more Gag-specific IFN-c secreting CD4+ T-cell precursors

Gag incorporation within the virus virion enhances CD4+ T-cell-induced effector function.

To investigate the effects of immunization of rVV-B5R/ Gag on CD4+ and CD8+ T-cell responses, we stimulated splenocytes with either class II- or class I-specific Gag peptides and quantitated the number of IFN-g producing cells by ELISpot. The cells producing IFN-g appeared as dark blue spots representing Gag-specific IFN-g producing effector cells. Mice vaccinated with rVV-B5R/Gag had significantly more Gag-specific IFN-g secreting CD4+ T cells than those from mice vaccinated with rVV-TK/Gag ( P < 0.0001) (Fig. 6A). In contrast, the number of Gagspecific CD8+ T cells making IFN-g was similar between the two groups ( P = 0.3) (Fig. 6B). Our data suggest that

rVV-B5R/Gag immunization produced more IFN-c in response to class II-specific Gag peptide We were also interested in whether the type of T-helper cell response differed between the groups of vaccinated mice. The production of IFN-g and IL-4 was measured as a correlate of Th1- vs. Th2-type cytokine responses, respectively. Ten days after immunization, splenocytes were harvested and incubated with Gag MHC class II or class I peptides and the supernatants were collected at various time points. CD4+ and CD8+ T cells produced significant levels of IFN-g, but very little IL-4, indicating a predominant Th1-

Fig. 6. Gag-specific IFN-g ELISpot assay of splenocytes from immunized mice. Groups of mice were immunized with rVV-B5R/Gag or rVV-TK/Gag or wildtype control virus and 10 days later splenocytes were plated (A, 5  105 splenocytes/well; B, 2.5  105 splenocytes/well) into IFN-g antibody-coated ELISpot plates with either (A) class II Gag peptide and stimulated overnight or (B) class I Gag peptide. Secreted IFN-g was detected and the number of colored spots was counted. Five mice were in each group per experiment and results were averaged from three separate experiments. Error bars represent standard deviation. rVV-B5R/Gag vs. rVV-TK/Gag: (A) P < 0.0001; (B) P = 0.3416. In the absence of antigen, 0.08% and 0.016% of cells were spontaneously secreting IFN-g in the CD4 and CD8 assay, respectively.

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Fig. 7. Gag-specific cytokine ELISA of splenocytes from immunized mice. Ten days after immunization, splenocytes were isolated and incubated with either (A) class II Gag peptide or (B) class I Gag peptide. At 6, 24, and 48 h after the incubation, supernatants were collected and production of IFN-g and IL-4 was measured by ELISA. Three mice were in each group per experiment and the experiment was carried out three separate times. The data shown are representative results. Open squares: rVV-B5R/Gag; open circles with dashed line: rVV-TK/Gag; closed triangle: wild-type virus. Error bars representing standard deviation were smaller than the symbols and thus do not appear on the figure.

type cytokine response (Fig. 7). Splenocytes from rVV-B5R/ Gag immunized group produced more IFN-g when stimulated with class II Gag peptide compared to the rVV-TK/Gag group (Fig. 7A). When cells were stimulated with class I Gag peptide, however, splenocytes from the rVV-TK/Gag group showed better IFN-g production at early time points (6 and 24 h) and similar IFN-g production at a later time point (48 h) (Fig. 7B).

Immunization with rVV-B5R/Gag protects mice in a heterologous challenge model To determine if the enhanced Gag-specific cellular immune responses elicited by rVV-B5R/Gag would correlate with improved protection, we challenged groups of vaccinated mice with a recombinant Listeria that expresses HIV-Gag (Lm-Gag). This experimental strategy of immu-

Fig. 8. Heterologous challenge of vaccinated mice with recombinant Listeria expressing HIV-Gag. Thirty days after vaccinia virus immunization, mice were challenged with a recombinant Listeria expressing HIV-Gag. Shown is data for colony counts in the homogenized spleen and liver from each mouse sacrificed on day 3 after Listeria challenge. For each set of points, the horizontal line represents the mean [rVV-B5R/Gag vs. rVV-control: P = 0.007 (for both the spleen and liver); rVV-B5R/Gag vs. rVV-TK/Gag: P = 0.03 and P = 0.007 (for the spleen and liver, respectively)].

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nization and heterologous challenge with an organism carrying the same recombinant antigen is similar to approaches used to test non-vaccinia virus-based vaccines (Belyakov et al., 1999; Mata et al., 2001; Rayevskaya and Frankel, 2001). Similar to our approach, this type of protection assay has also been specifically used to study vaccinia vaccination followed by Listeria challenge (An et al., 1996). Thirty days after a single vaccinia virus immunization, groups of mice were challenged with 0.2 LD50 of Lm-Gag. At various times after challenge, groups of mice were sacrificed and the colony counts of Listeria were determined in the spleen and liver. At day 3 post challenge (Fig. 8), there were significantly lower colony counts in the spleen and liver of mice immunized with rVV-B5R/ Gag when compared to mice.

Discussion Improving the immunogenicity of poxvirus vaccine vectors is important when considering issues of safety and efficacy of such vaccines. One approach to improve immune responses to foreign antigens, which has not been extensively explored with poxviruses, is to manipulate the site of foreign antigen expression. To date, the majority of poxvirus-based vaccines produce antigens that either remain in the cytoplasm, are secreted from infected cells, or are expressed on the outer membrane of infected cells. Directed incorporation of foreign antigens into the vaccinia virion is distinct from the inclusion on the virion of some host antigens that has been identified to happen during viral morphogenesis (Gomez and Esteban, 2001; Krauss et al., 2002; Vanderplasschen et al., 1998). Our knowledge of the structure –function relationship of domains of the EEVspecific B5R protein allowed us to construct a recombinant virus that specifically incorporated a foreign antigen into EEV. We made the recombinant viruses in a strain of vaccinia virus (i.e., IHDJ) that releases relatively high amounts of EEV (Payne, 1980). The resulting recombinant virus (rVV-B5R/Gag) made smaller plaques under semisolid overlay and released less EEV than wild type. This result differs from the studies using a recombinant vaccinia virus that fused GFP to the cytoplasmic tail of B5R and made plaques similar in size to the wild-type virus (Ward and Moss, 2001). The differences in plaque size between these two recombinant viruses may have multiple explanations. The Gag protein that we fused to B5R is about twice the size of GFP. Alternatively, there may be protein – protein interactions between Gag fused to B5R. Finally, as opposed to the GFP construct, our construct deletes the cytoplasmic tail. Antigen expression on particles or virions has been explored in other systems (Allsopp et al., 1996; Casal, 1999; Evans et al., 1989; Mandl et al., 1998; Ulrich et al., 1998). Antigens presented on particles may result in enhanced immunity for several reasons. HIV-envelope dis-

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played on EEV (Katz and Moss, 1997) may enhance the humoral immune response due to its presentation as a particulate, polyvalent form, rather than a monomeric protein (Ulrich et al., 1998). We hypothesized that directly incorporating a foreign antigen into a vaccinia virion would improve the cellular immune response by allowing released virions containing the antigen to be directly taken up by antigen-presenting cells (APC). In such a form, the APC could begin to directly process the foreign antigen that was engulfed as part of the virion resulting in augmented Thelper cell responses. In addition, delivery of an antigen in the context of a virion might supply the necessary ‘‘danger’’ signal required to produce an antigen-specific immune response (Gallucci and Matzinger, 2001). The released virions could also produce newly synthesized antigen within the APC, similar to standard vaccinia virus vaccine vectors. Our findings of improved CD4-helper responses and enhanced protection in a heterologous challenge model support this hypothesis. Although HIV-Gag expressed by itself is known to self-assemble into particles (Gheysen et al., 1989; Karacostas et al., 1989), we did not include a portion of the N-terminus of Gag in the recombinant viruses to compare the immune responses to a foreign antigen specifically incorporated into EEV with a standard recombinant vaccinia virus that did not incorporate the foreign antigen into the virion (or release foreign antigen particles). Because a dramatic loss of CD4+ T cells is associated with HIV infection, our finding that rVV-B5R/Gag elicits enhanced Thelper cell responses (with a Th1 bias) may aid in designing preventative and therapeutic vaccines against HIV. Furthermore, because the recombinant virus that expresses a chimeric B5R protein forms smaller plaques and has slightly altered growth characteristics in tissue culture, it may have an improved safety profile when compared to a standard vector. Because the test construct and the standard recombinant vaccinia virus vector were genetically different (i.e., they had differences in their TK and B5R genes), a concern might be that the enhanced Gag-specific immune response to rVV-B5R/Gag (mutated B5R gene/TK+) was due to that virus being a better overall immunogen than rVV-TK/Gag (wild-type B5R gene/TK ). However, we found that vaccinia virus-specific immune responses to each recombinant virus were essentially identical. Another potential concern is that because the two viruses under study express Gag from different promoters, the rVV-B5R/Gag may be expressing more protein and thus provide a better immune response to the antigen (Hemmer et al., 1998; Wherry et al., 1999). However, although rVV-B5R/Gag induces a more potent immune response to Gag, this recombinant synthesized lower levels of antigen than the standard recombinant vector (rVV-TK/Gag). The strong synthetic early/late promoter (Chakrabarti et al., 1997) used to express Gag in the rVV-TK/Gag results in the production of more protein than the natural B5R promoter used by rVV-B5R/Gag. In total, these findings support our

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conclusion that targeting a foreign protein to be incorporated into EEV enhances the immune response to the foreign protein when compared to a standard recombinant vaccinia virus. Because primary CD8+ T-cell responses were similar between rVV-B5R/Gag and the standard recombinant vector (rVV-TK/Gag), the protection seen after Listeria challenge may indicate that CD4+ T-cell responses are important for the control of Listeria infection. The role of CD4+ T cells in controlling Listeria infections is somewhat controversial. Some studies suggest that they play no role in protection against Listeria challenge (Baldridge et al., 1990; Mielke et al., 1988, 1989), whereas others suggest that both CD4+ and CD8+ T cells are important (Kaufmann et al., 1985; Ladel et al., 1994; Sasaki et al., 1990). There are also many studies claiming that CD4+ T cells alone can mediate clearance of Listeria (Bhardwaj et al., 1998; Magee and Wing, 1988; Rakhmilevich, 1994; Sasaki et al., 1990). CD4+ T-cellmediated clearance of Listeria may be possible for lowchallenge doses with Listeria, but CD8+ T cells must play a critical role in clearing fulminant infections, because, in addition to infecting MHC class II positive cells such as macrophages and other APCs, Listeria also infects class II negative cells such as hepatocytes. In an attempt to demonstrate that CD4+ T-cell responses are important for the control of Listeria infection in our system, before Listeria challenge, we depleted CD4+ or CD8+ cells from groups of mice using standard protocols (Mata et al., 2001). However, the isotype control antibody altered the Listeria counts in control animals thus making it difficult to interpret data from vaccinated animals that are then depleted. The effect the control antibody had on Listeria infection may have been caused by nonspecific macrophage activation that facilitated early control of the Listeria infection.

Materials and methods Plasmid constructs used to generate recombinant viruses We constructed a transfer plasmid (pSI-169g) that enabled in-frame cloning of Gag into the B5R ORF that replaces the B5R cytoplasmic tail. The plasmid contains the natural B5R promoter region for early/late expression (Engelstad et al., 1992) of the chimeric protein, the natural B5R signal peptide (Isaacs et al., 1992) to direct the chimeric protein to the secretory pathway, flanking vaccinia virus sequences that directs insertion into the B5R region of vaccinia virus by homologous recombination (Earl and Moss, 1991), and an efficient way to isolate recombinant viruses by a positive selection procedure (Isaacs et al., 1990). A fragment of Gag from the HXB2 clone of prototype HIV strain 3B (from amino acid 29 to 498) was generated by PCR using appropriate primers that allowed in-frame cloning into the pSI-169g. We chose this portion of Gag because it correlated exactly with the segment

expressed by a recombinant Listeria expressing HXB2 Gag (Mata et al., 2001) that we used to challenge vaccinated mice. Sequencing of the resulting plasmid (pSI-171k) confirmed that no inadvertent mutations were introduced and transient transfection of vaccinia virus-infected cells confirmed that the chimeric protein was produced from pSI171k. To generate a standard vaccinia virus expressing HIVGag, we cloned the identical HXB2 Gag fragment into pSC65 (Chakrabarti et al., 1997). The resulting plasmid (pSI-172-4) allows recombination into the vaccinia virus thymidine kinase gene (TK) and the expression of the cloned Gag to be driven by a vaccinia virus strong synthetic early/late promoter. Isolation of recombinant vaccinia viruses and characterization Recombinant viruses were generated by homologous recombination in cells infected with a B5R deletion virus (vSI-15) and transfected with p171k. vSI-15 was a previously isolated vaccinia virus (strain IHDJ) in which the majority of the B5R ORF was replaced with a gpt selection cassette (Wolffe et al., 1993). The recombinant virusexpressing Gag fused to B5R (vSIAA-32; called rVVB5R/Gag in this paper) was isolated and plaque purified by reverse gpt selection (Isaacs et al., 1990). The standard recombinant vaccinia virus-expressing Gag was generated by homologous recombination in cells infected with IHDJ and transfected with pSI-172-4. This virus (vSIAA-34; called rVV-TK/Gag in this paper) was isolated and plaque purified following standard techniques and plaque isolation on a TK- cell line in the presence of bromo-deoxyuridine and x-gal (Earl and Moss, 1991). Western blots of infected cell lysates showed that Gag is expressed and recognized by a rabbit polyclonal antiserum to Gag, HIV-1 p25/24 Gag antiserum (Steimer et al., 1986) (data not shown). Western blot analysis of the Gag protein present in EEV was performed on unlabeled virus in the media of infected cells that was pelleted through a 36% sucrose cushion. The amount of Gag produced by each virus was quantitated using a P24 assay on lysates of virus infected cells. Briefly, BSC-1 cells were infected with each virus at an m.o.i. of 1 and incubated for 20 h at 37 jC in 5% CO2. The cells were harvested, centrifuged, and resuspended in 0.1% Triton X100. p24 Gag levels on diluted specimens were determined by p24 ELISA (Coulter, Krefeld, Germany) according to the manufacturer’s instructions. Virus growth kinetics were measured on confluent wells of BSC cells infected at m.o.i. of 5 pfu/cell and 0.005 pfu/cell with rVV-B5R/Gag, rVV-TK/Gag, or wild-type virus in duplicate. At various time points, media and cells were harvested and virus titered. To confirm that EEV was indeed produced by the recombinant virus expressing a chimeric B5R-Gag protein, we carried out CsCl gradient centrifugation analysis on metabolically labeled virus released from infected cells into the media (Payne and Norrby, 1976).

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Mice and immunizations Female BALB/c (H-2d) mice were purchased from Charles River Laboratories (Raleigh, NC) and maintained in a pathogen-free microisolator environment. Mice used in this study were 6 – 12 weeks old. Six- to eight-week-old female BALB/c mice were immunized once by intraperitoneal inoculation with approximately 2  106 pfu of recombinant vaccinia viruses or wild-type virus. Although tail scarification is frequently a method of vaccinating mice, we chose to use the intraperitoneal route because it allowed us to use a lower dose of virus than is often used with tail scarification and it made us more confident that the mice were receiving the targeted dose of virus. Because we were interested in examining the primary immune response (and not a memory response), we sacrificed mice 10– 14 days after vaccination. Vaccinia-specific immune responses All assays examining vaccinia-specific immune responses were performed with splenocyte suspensions prepared from mice immunized 2 weeks earlier with rVV-B5R/Gag, rVVTK/Gag, or WT virus. Cells were enriched for lymphocytes by plating in 6-well plates at 2  106 cells/well, incubating at 37 jC, and removing the non-adherent cells after 3 h. For vaccinia-specific cytokine production, cells were resuspended in complete RPMI and IFN-g ELISpot was performed using an IFN-g ELISpot kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Briefly, 2  105 splenocytes were plated into each well and co-cultured with 1  105 or 5  104 antigenpresenting cells (APCs). APCs were naı¨ve splenocytes infected for 1 h at a ratio of 3 pfu/cell with vaccinia virus (strain WR) and irradiated. Uninfected APCs were used to quantitate nonspecific responses. After 24 h, wells were washed and plates were incubated overnight with a biotinylated anti-IFN-g mAb. IFN-g-producing cells were detected with alkaline phosphate-streptavidin and BCIP/NBT chromogen substrate. Positive cells were quantitated using a dissection microscope. For vaccinia-specific cell proliferation, cells were resuspended in complete RPMI and 1  106 cells were plated in 96-well flat-bottomed plates and co-cultured with 5  105 restimulator cells. After 48 h, proliferation was measured by BrdU incorporation using a cell proliferation ELISA kit (Roche Diagnostics, Indianapolis, IN). Briefly, splenocytes were incubated for 2 h with BrdU labeling solution. Cells were then fixed, and proliferating cells detected using antiBrdU-peroxidase and tetramethyl-benzidine (TMB) substrate. Absorbance was measured using an ELISA reader at 370 nm. For vaccinia-specific cytotoxic CD8+ T lymphocyte responses, two-thirds of each spleen from vaccinated mice were resuspended in complete RPMI and cultured in 75-cm tissue culture flasks at 37 jC. The remaining third was

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resuspended in 5% FBS and infected at a ratio of 3 pfu/cell with WR. After 1 h, the infected restimulators were added to the cultured flasks. After 6 days, the cells were harvested and plated in 96-well round-bottomed plates at 1  106 cells/well. Four 2 dilutions were made for each sample. Cells were co-cultured with 2  104 target cells. Target cells were P815 cells infected for 1 h at a ratio of 5 pfu/cell with WR and labeled with Calcein AM (Molecular Probes, Eugene, OR). Uninfected targets were used as controls. After 3 h, plates were centrifuged and the supernatant transferred into 96-well flat-bottomed Fluoro Nunc plates. Calcein release was quantitated using a fluorometer at 538 nm emission and 485 nm excitation. For anti-vaccinia virus antibody responses, 96-well flatbottomed ELISA plates were coated overnight at 4 jC using whole cell vaccinia-infected cell lysates (Hammarlund et al., 2003) in bicarbonate coating buffer. Briefly, 48 h after infection with WR, infected HeLa cells were harvested, spun down, and lysed in RIPA buffer. This material was then diluted 1:100 in bicarbonate coating buffer and added to plates. Serial 3-fold dilutions of sera, harvested from naı¨ve mice or groups of mice immunized 2 weeks earlier, were incubated for 1 h at room temperature. Plates were washed and incubated with horseradish peroxidase-conjugated rabbit anti-mouse IgGAM (1:2000; Zymed, San Francisco, CA). After 1 h, samples were detected with OPD (Acros Organics, NJ). Reaction was stopped with 1 M HCl and absorbance measured using an ELISA reader at 490 nm. Peptides All of the T-cell epitopes for the H-2d mouse used in this study have been previously defined. The Kd-restricted class I Gag peptide is sequence 197 –205 (AMQMLKETI) (Mata et al., 1998) and the Kd-restricted control peptide is from Listeriolysin O (LLO), sequence 91 – 99 (GYKDGNETI) (Pamer et al., 1991). The H-2d restricted class II Gag peptide is sequence 253 – 272 (NPPIPVGEIYKRWIILGLNK) (Mata and Paterson, 1999). The control MHC class II binding peptide was the pigeon cytochrome c epitope (KAERADLIAYLKQATAK) (Schwartz, 1985). Peptides Gag 197 –205 and LLO 91 –99 were purchased as HPLC purified products from the Biopolymer Synthesis Center, California Institute of Technology, Pasadena, CA. All other peptides were synthesized in our laboratory using DuPont’s RaMPS multiple peptide synthesis system procedure on Wang Resin ( p-Alkoxy-benzyl Alcohol Resin) (DuPont Chemical Company) using Fmoc (9-Fluorenylmethyloxycarbonyl) nitrogen terminal protected amino acids (BACHEM Biotech Company, Philadelphia, PA). Couplings were carried out using DPC (N-N-diisopropylcarbodiimide), HOBT (1-Hydroxybenzotriazole) in DMF (N,N-dimethylformamide) solvent and were monitored by ninhydrin reaction in 100 jC water for 5 min. Simultaneous resin cleavage and side-chain deprotection were achieved by high-concentration TFA (all chemical reagents purchased from Aldrich

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and Sigma Company). All crude products were purified to 98% purity by reverse-phase high performance liquid chromatography (HPLC) (Perkin Elmer Series 400) using a Vydac C18 column and their molecular weight verified by MALDI-Mass Spectroscopy. Gag-specific proliferation assay Ten days after the primary immunization, groups of mice were sacrificed and spleens were harvested. Spleens were processed through nylon mesh and single cell suspensions were prepared. Red blood cells were lysed and splenocytes were resuspended in complete RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, 1 mM sodium pyruvate, and 50 AM hmercaptoethanol. A total of 5  105 cells were plated into 96well microplates in triplicate with class II Gag peptide (10 AM final concentration) and incubated for 48 h. As controls, 10 AM of pigeon cytochrome c peptide or 1 Ag/ml of con-A were added. Proliferation was measured by colorimetric assay 4 h after adding WST-1 reagent (Roche Diagnostics, Germany). This assay reflects the metabolic activity of a proliferating cell population and when compared to BrdU and 3Hthymidine incorporation assays gave the same results (Drew Weissman, University of Pennsylvania; personal communication). O.D. was measured at 450 nm. Stimulation index (S.I.) was calculated as O.D. with peptide/O.D. without peptide. Gag-specific IFN-c ELISpot Splenocytes were prepared from immunized mice and resuspended in complete RPMI and counted. IFN-g ELISpot was performed using murine IFN-g ELISpot kit (R&D Systems) following the manufacturer’s instructions. Briefly, 2.5  105 or 5  105 splenocytes were plated into each well in duplicate and incubated with 1 AM of Gag class I epitope, 10 AM of Gag class II epitope, or control peptides at 37 jC. Control peptides were either LLO 91– 99 (1 AM) or pigeon cytochrome c (10 AM). After 24 h, wells were washed and secreted IFN-g was incubated with biotinylated anti-IFN-g antibody, followed by alkaline phosphatase-conjugated streptavidin and substrate, BCIP/NBT chromogen. Plates were washed, dried, and colored spots were counted in blinded fashion using dissection microscope. The numbers of spots (approximately 40) from splenocytes immunized with wild-type virus were subtracted from those immunized with each recombinant virus. Gag-specific cytokine ELISA Splenocytes (6  106 cells/well) from immunized mice were prepared and incubated with either class I Gag peptide (1 AM/well) or class II Gag peptide (10 AM/well), for 6, 24, and 48 h. At each time point, supernatants were collected and the concentrations of IFN-g and IL-4 in supernatants were

measured using mouse IFN-g and IL-4 ELISA kits (eBioscience, San Diego, CA) following the manufacturer’s instructions. Briefly, plates were coated with capture antibody overnight, blocked with assay diluent, and washed with wash buffer three times. Standards and experimental supernatants were added to appropriate wells in duplicate. Two hours later, plates were developed with avidin-HRP and TMB as a substrate and were read at 450 nm. The levels of IFN-g and IL-4 were calculated by comparison with a standard curve using murine IFN-g and murine IL-4. The lower limit of detection for the assay was approximately 4 pg/ml. Mouse challenge studies To examine if vaccinated mice had different responses to vaccinia virus challenge, groups of 6– 7 weeks old BALB/c mice were immunized intraperitoneally with rVV-B5R/Gag, rVV-TK/Gag, or WT virus. Four weeks after immunization, mice were challenged intranasally with 2  106 pfu of WR. Mice were weighed before and each day following infection and percentage of weight loss was calculated over time. To determine if vaccinated groups could control a heterologous challenge of an organism expressing HIV-Gag, we used a previously described Listeria monocytogenes expressing HIV-Gag (Lm-Gag) (Mata et al., 2001). Groups of 6- to 7-week-old female BALB/c mice were immunized intraperitoneally with approximately 2  106 pfu of the various recombinant or wild-type viruses. Approximately 30 days after immunization, mice were challenged intraperitoneally with 1  106 cfu of Lm-Gag. At 3, 6, and 10 days, mice were sacrificed and the liver and spleen were harvested. Listeria colony counts were determined after homogenization of the tissue and plating of serial dilutions of the homogenate on BHI agar. Colonies were only detectable on day 3. At later time points, few colonies were detectable in the liver. Statistical analysis Statistical differences between groups were determined with the Mann –Whitney test and Student’s t test.

Acknowledgments This work was supported in part by a University of Pennsylvania Center for AIDS Research (CFAR) developmental pilot project core and by NIH grant number AI47237 to S.N.I. and NIH grant number AI 36657 to Y.P. Antiserum to Gag (HIV-1 p25/24 Gag) was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. We also would like to thank Dr. Yoon Ahn for help with the construction of the transfer plasmid p169g, Edward Bell for technical assistance, and Penn’s CFAR Virus, Cell and Molecular Core for the P24 assay.

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