HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

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INTRODUCTION

According to UNAIDS, about 36.7 million adults and children were living with human immunodeficiency virus (HIV) worldwide at the end of 2015; however, the number of people newly infected continues to fall, being 38% lower in 2015 than in 2001 (http://www.unaids.org/). These data are the result of the global implementation of preventive and therapeutic strategies. Nevertheless, the development of a vaccine remains among the best hopes for controlling the HIV/AIDS pandemic.

To date, the phase III clinical trial RV144 is the only HIV-1 vaccine efficacy trial that has demonstrated a modest level of protection (31.2%) against HIV-1 infection in humans (1). The RV144 study combined a recombinant canarypox virus vector (ALVAC) expressing HIV-1 antigens from clades B and E as a prime with recombinant HIV-1 gp120 proteins from clades B and CRF01_AE as a boost. Further studies evaluating potential immune correlates of protection have shown that CD4+ T cells, IgG antibodies to the V1/V2 and V3 loops of HIV-1 gp120, and IgG3 antibodies to gp120, together with antibody-dependent cellular cytotoxicity (ADCC) responses, correlated with a decreased risk of HIV-1 infection, whereas IgA antibodies to the envelope protein correlated with decreased vaccine efficacy in the vaccine group (2,–7). These clinical findings for the first time provided evidence that an HIV/AIDS vaccine can prevent HIV-1 infection and highlighted that poxvirus vectors should be considered one of the future HIV/AIDS vaccine candidate vectors.

Among the poxviruses, the highly attenuated vaccinia virus (VACV) strain NYVAC has emerged as a potential HIV/AIDS vaccine candidate (8, 9). Importantly, several NYVAC-based recombinant viruses expressing HIV-1 antigens from clade B or C have been evaluated as HIV/AIDS vaccine candidates in preclinical (10,–28) and clinical (20, 29,–34) studies, with encouraging results for HIV-1-specific T cell and humoral immune responses. However, new strategies have been implemented to improve poxvirus vector immunogenicity (35). Among these strategies, we previously reported that the deletion of immunomodulatory VACV genes such as B19R and/or B8R, which block interferon (IFN) type I and type II pathways (17), or the insertion of the host range VACV C7L gene into NYVAC recombinant vectors expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens as a polyprotein (NYVAC-C) significantly improved the magnitude and quality of HIV-1-specific immune responses in mice (23). Furthermore, the deletion of the B19R and/or B8R gene in NYVAC-C triggered an upregulation of innate immune pathways in infected human monocytes, with robust expression of type I IFNs and IFN-stimulated genes (ISGs), strong activation of the inflammasome, and an upregulation of the expression of interleukin-1β (IL-1β) and proinflammatory cytokines (12). Moreover, the restoration of replication competence of NYVAC-C in human cells by the reincorporation of the K1L and C7L VACV host range genes (NYVAC-C-KC) with or without the removal of the immunomodulatory viral molecule B19 enhanced the cross-presentation and proliferation of HIV-1-specific memory CD8+ T cells in vitro (26). These recombinant vectors selectively activated IFN-induced genes and genes involved in antigen processing and presentation, as determined by microarray analysis of infected human dendritic cells (DCs) (19, 26). At the same time, these constructs maintained limited virus spread in tissues and an attenuated phenotype (26). Additionally, further improved NYVAC recombinant vectors expressing HIV-1 immunogens, such as HIV-1 clade C(ZM96) trimeric soluble gp140 or Gag(ZM96)-Pol-Nef(CN54) as Gag-derived virus-like particles (VLPs), have been shown to have an enhanced HIV-1-specific immunogenicity profile in mice (24) and nonhuman primates (NHPs) (10, 13).

Clinical trials with homologous NYVAC vectors expressing HIV-1 antigens (gp120 Env and the polyprotein Gag-Pol-Nef) have shown a limited immunogenicity profile with a preference for CD4+ T cell activation, which was markedly enhanced when priming was performed with a DNA vector expressing the same HIV-1 antigens (29,–33). Thus, in order to optimize the immunization protocol with NYVAC vectors expressing HIV-1 antigens, various approaches in NHPs have been evaluated, either comparing NYVAC to ALVAC (13) or combining NYVAC with DNA vectors (10), peptides (22), and dendritic cell targets (28), which have all demonstrated promising results.

Here, as part of the Poxvirus T Cell Vaccine Discovery Consortium (PTVDC) from the Collaboration for AIDS Vaccine Discovery (CAVD) of the Bill and Melinda Gates Foundation, we extended our previous studies with NYVAC recombinant vectors (19, 26) and evaluated novel NYVAC recombinant vectors in NHPs. Hence, using a single recombinant NYVAC vector, we combined a set of strategies: restoration of replication competence, expression of novel HIV-1 immunogens (trimeric gp140 and Gag-Pol-Nef as Gag-derived VLPs), and deletion of the B19R immunomodulatory gene (NYVAC recombinant vectors termed NYVAC-C-KC and NYVAC-C-KC-ΔB19R). Thus, NYVAC-C-KC and NYVAC-C-KC-ΔB19R were compared in immunized NHPs to evaluate the HIV-1-specific immunogenicity profile induced by these novel NYVAC recombinant vectors when applied in a prime-boost approach according to a protocol for the delivery of the immunogens, poxvirus, and protein similar to the protocol used in the RV144 phase III clinical trial. The aim was to define the type of HIV-1-specific T cell and humoral immune responses induced by these vectors as a function of immunological markers that have been correlated with HIV-1 immune efficacy. The results showed that replicating NYVAC-C-KC vectors together with a booster of the purified gp120 protein component induced an enhanced activation of HIV-1-specific CD4+ and CD8+ T cell immune responses, together with a strong induction of HIV-1-specific humoral immune responses. These results demonstrate that replicating NYVAC-C-KC vectors triggered relevant HIV-1-specific immunological properties as potential correlates of protection, with the VACV B19 protein exerting some control of immune functions and supporting the use of these novel NYVAC-C-KC recombinant vectors as HIV/AIDS vaccine candidates.

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RESULTS

 

Enhanced expression, plaque size, and innate immune profile of NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors.

We previously described the generation and characterization of nonreplicating NYVAC vectors expressing clade C HIV-1 trimeric gp140 or Gag-Pol-Nef as a polyprotein processed into Gag-derived VLPs and their immune behavior in mice (24) and in NHPs (13). Since these vectors do not replicate in human cells, it was important to define whether novel replication-competent NYVAC-KC vectors could be more immunogenic as a function of higher levels of antigen expression during infection. Analysis of the expression of HIV-1 gp140 in human HeLa cells by Western blotting is shown in Fig. 1A. Clearly, higher levels of expression at late times postinfection were observed in the NYVAC-C-KC vectors than those of the parental NYVAC-C vector. These differences were also noticeable after analysis of the virus plaque size phenotype in cultured BSC-40 cells. The NYVAC-C-KC vectors have a larger plaque size, with or without B19R, than did the parental wild-type NYVAC (NYVAC-WT) or NYVAC-gp140 vector, consistent with a higher replication capacity of NYVAC-KC vectors than of parental NYVAC (Fig. 1B). Analysis of the innate immune response elicited in human macrophages (THP-1) infected with the NYVAC-C-KC vectors by real-time PCR (RT-PCR) showed that compared to NYVAC-KC-gp140, NYVAC-KC-gp140-ΔB19R triggered a significant upregulation of the mRNA levels of type I IFN (IFN-β), macrophage inflammatory protein 1α (MIP-1α), IL-8, and IL-1β (Fig. 1C), indicating some differential innate immune responses between these vaccines.

 

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FIG 2

Immunization schedule for nonhuman primates. (A) Immunization groups included in the AUP513 study. Eight NHPs (rhesus macaques) in each group were immunized at weeks 0 and 4 with the corresponding replication-competent NYVAC-C-KC poxvirus vector (NYVAC-C-KC or NYVAC-C-KC-ΔB19R) and at weeks 12 and 24 with a combination of the replication-competent NYVAC-C-KC poxvirus vector plus a clade C HIV-1 gp120 protein, as detailed in Materials and Methods. The compositions of NYVAC-C-KC, NYVAC-C-KC-ΔB19R, and the bivalent clade C HIV-1 gp120 protein are detailed in Materials and Methods. (B) Chronological diagram showing the immunization schedule and the immunogenicity endpoints used in this study. At weeks 0, 4, 12, and 24, animals were immunized as described above for panel A. A dose of 1 × 108 PFU of each recombinant replication-competent poxvirus vector (NYVAC-KC-gp140 plus NYVAC-KC-Gag-Pol-Nef for the NYVAC-C-KC vector and NYVAC-KC-gp140-ΔB19R plus NYVAC-KC-Gag-Pol-Nef-ΔB19R for the NYVAC-C-KC-ΔB19R vector; 2 × 108 PFU of total virus) and 50 μg of each clade C HIV-1 gp120 protein (TV1 gp120 plus 1086 gp120; 100 μg of total protein) were used for each immunization. The bivalent clade C gp120 protein was administered together with the MF59 adjuvant. At weeks 0, 6, 14, 26, and 36 (at the beginning of the study; 2 weeks after the second, third, and fourth immunizations; and at the end of the study, respectively), PBMCs, serum, and rectal mucosal samples were obtained from each immunized animal, and HIV-1-specific T cell and humoral immune responses were analyzed.

 

HIV-1-specific T cell immune responses.

The total magnitude of HIV-1-specific T cell immune responses induced by the NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors was measured at weeks 0, 6, 14, 26, and 36 by an IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay. The results showed that the mean spot-forming unit (SFU) values induced in both immunization groups were low until week 14 and clearly peaked at week 26 (2 weeks after the completion of the prime-boost immunization protocol) (Fig. 3A). As expected, the SFU values declined at week 36. There were no significant statistical differences in the levels of IFN-γ-positive T cells between the two groups at any time point. However, at week 26 of immunization, NYVAC-C-KC elicited a trend toward higher numbers of SFUs, while at week 36 of immunization, NYVAC-C-KC-ΔB19R induced a trend toward higher responses.

 

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FIG 3

Magnitude of HIV-1-specific T cell immune responses. PBMCs were collected from each rhesus macaque (n = 8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R at weeks 0, 6, 14, 26, and 36, and the total magnitude of HIV-1-specific T cell immune responses triggered by the different immunization regimens were measured by ELISpot (A) or ICS (B and C) assays following stimulation of PBMCs with HIV-1 Env, Gag, Pol, and Nef peptide pools. (A) Total magnitudes of SFUs per million cells, with values representing the number of IFN-γ-positive T cells against Env, Gag, Pol, and Nef peptide pools. (B and C) Total magnitudes of HIV-1-specific CD4+ (B) and CD8+ (C) T cell responses elicited in the different immunization groups, with the values representing the sums of the percentages of T cells producing IFN-γ, and/or TNF-α, and/or IL-2 against Env, Gag, Pol, and Nef peptide pools. Values for unstimulated controls were subtracted in all cases. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. P values and the numbers of responding rhesus macaques are indicated.

Furthermore, we also analyzed the magnitude of HIV-1-specific T cell immune responses induced by the NYVAC-C-KC and NYVAC-C-KC-ΔB19R vectors by an intracellular cytokine staining (ICS) assay. Thus, at weeks 6, 14, 26, and 36, we measured the percentages of HIV-1-specific CD4+ and CD8+ T cells secreting IFN-γ, IL-2, and/or tumor necrosis factor alpha (TNF-α) after stimulation of PBMCs obtained from each immunized rhesus macaque with peptide pools that spanned the HIV-1 Env, Gag, Pol, and Nef antigens present in the inserts. As shown in Fig. 3B and ​andC,C, the total magnitude of HIV-1-specific CD4+ and CD8+ T cell responses peaked at week 26, with a decline at week 36, similar to the results obtained with the IFN-γ ELISpot assay. There were no statistically significant differences in the magnitudes of HIV-1-specific CD4+ T cell responses induced in both immunization groups at any time point (Fig. 3B). However, at weeks 14 and 36 of immunization, NYVAC-C-KC-ΔB19R induced a trend toward higher HIV-1-specific CD4+ T cell responses, while at week 26 of immunization, NYVAC-C-KC induced a trend toward higher responses. On the other hand, the analysis of the magnitude of the total HIV-1-specific CD8+ T cell responses (Fig. 3C) showed that immunization with NYVAC-C-KC-ΔB19R induced a trend toward increased levels over NYVAC-C-KC at all time points, but the differences were not significant.

Moreover, the analysis of the cytokine responses generated by both immunization groups revealed that in agreement with the total magnitude of HIV-1-specific CD4+ T cell responses, at week 36, immunization with NYVAC-C-KC-ΔB19R induced a trend toward higher magnitudes of responses of HIV-1-specific CD4+ T cells producing TNF-α (Fig. 4B) or IL-2 (Fig. 4C), but the differences were not significant. However, at week 26 of immunization with NYVAC-C-KC, a trend toward higher magnitudes of responses of HIV-1-specific CD4+ T cells producing IFN-γ (Fig. 4A), TNF-α (Fig. 4B), or IL-2 (Fig. 4C) was observed.

 



 

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FIG 5

Binding IgG antibody responses. (A to D) Total plasma binding IgG antibody levels against the HIV-1 gp140 group M consensus (sCon) (A), gp120 from isolate TV1 (B), gp120 from isolate 1086 (C), and MuLV gp70-scaffolded V1/V2 proteins (D) induced by the different immunization groups. (E) Rectal binding IgG antibody levels against the HIV-1 gp140 group M consensus and gp120 proteins from isolates 1086 and TV1 induced in the different immunization groups. Individual serum or rectal mucosal samples were obtained from each rhesus macaque (n = 8 per group) immunized with NYVAC-C-KC or NYVAC-C-KC-ΔB19R at weeks 6, 14, 26, and 36. Binding IgG antibodies were measured by a BAMA (A to D) or by analyzing the binding magnitude normalized to total rhesus IgG (specific activity) (E), as indicated in Materials and Methods. The magnitudes of the total binding IgG antibody responses are expressed as the AUC from serial dilutions (Dil) of plasma. Each dot represents the value for one immunized macaque. Box plots represent the distribution of data values, with the line inside the box indicating the median value. P values and the numbers of responding rhesus macaques are indicated.

Similarly to the results shown in Fig. 5, cross-clade binding IgG antibody levels induced in both immunization groups were low by week 6 but increased markedly by week 26 and waned with time at week 36 but remained at levels similar to or higher than those at week 6 (data not shown). Overall, both immunization groups induced similar high levels of cross-clade binding IgG antibodies against consensus HIV-1 gp140 proteins of clade A and clade B, clade A gp140(00MSA), and clade B gp140(JRFL), with no statistically significant differences between the groups. A trend toward higher levels of cross-clade binding IgG antibodies for NYVAC-C-KC was noted at week 14.

In addition, rectal IgG binding responses against the group M HIV-1 gp140 consensus and clade C gp120 proteins from isolates 1086 and TV1 were detected in both immunization groups, with a peak response at week 26 and a decline at week 36 (Fig. 5E), although differences were not significant. The rates of responders at week 26 were higher in the NYVAC-C-KC-ΔB19R immunization group, while at week 36, the rates were higher in the NYVAC-C-KC immunization group.

Because the RV144 phase III clinical trial showed that high levels of binding plasma IgA antibodies to HIV-1 Env correlated directly with an increased risk of infection (4, 5), we next analyzed the binding IgA antibodies induced after immunization with the two NYVAC-C-KC vectors in serum and in rectal mucosa (data not shown). The results for serum showed that very low levels (barely above the background level for most time points) of binding IgA antibodies against the consensus HIV-1 gp140 protein of clade C, gp120 from isolate TV1, and the MuLV gp70-scaffolded protein were induced in both immunization groups. Furthermore, similar negligible levels of binding IgA antibodies to consensus HIV-1 gp140 proteins of clades A and B and group M, clade A gp140(00MSA), and clade B gp140(JRFL) were observed for the two vaccinated groups. There were no rectal IgA binding antibodies (data not shown).

 

ADCC responses.

Since ADCC can mediate nonneutralizing antibody effector functions dependent on Fc receptor engagement and was suggested to be a correlate of protection in the RV144 phase III clinical trial (4, 36), we next analyzed the ability of NYVAC-C-KC and NYVAC-C-KC-ΔB19R to induce ADCC responses against HIV-1 gp120 proteins from isolates TV1 and 1086 at weeks 0, 26, and 36 in individual serum samples obtained from each immunized rhesus macaque (Fig. 6A). The results showed that both immunization groups elicited similar titers of ADCC against HIV-1 gp120 from isolates TV1 and 1086 at week 26, with a decline at week 36. The titers against target cells coated with the HIV-1 gp120 protein of the TV1 isolate were higher than those obtained with HIV-1 gp120 of the 1086 isolate. Although there were no statistical differences between plasma samples from animals immunized with the two NYVAC-C-KC vectors, immunization with NYVAC-C-KC-ΔB19R induced a trend toward higher ADCC titers against the HIV-1 gp120 protein of the TV1 isolate at week 36 than with NYVAC-C-KC. On the other hand, NYVAC-C-KC elicited higher-level ADCC responses against HIV-1 gp120 from isolate 1086.

 

Jawahar Raina

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