The encouraging results of the RV144 vaccine trial have spurred interest in poxvirus prime-protein boost human immunodeficiency virus (HIV) vaccine modalities as a strategy to induce protective immunity. Because vaccine-induced protective immunity is critically determined by HIV envelope (Env) conformation, significant efforts are directed toward generating soluble trimeric Env immunogens that assume native structures. Using the simian immunodeficiency virus (SIV)-macaque model, we tested the immunogenicity and efficacy of sequential immunizations withDNA(D), modified vaccinia virus Ankara (MVA) (M), and protein immunogens, all expressing virus-like particles (VLPs) displaying membrane-anchored trimeric Env. A single VLP protein boost displaying trimeric gp160 adjuvanted with nanoparticle-encapsulated Toll-like receptor 4/7/8 (TLR4/7/8) agonists, administered 44 weeks after the secondMVA immunization, induced up to a 3-fold increase in Env-specific IgG binding titers in serum and mucosa. Importantly, the VLP protein boost increased binding antibody against scaffolded V1V2, antibody-dependent phagocytic activity against VLP-coated beads, and antibody breadth and neutralizing antibody titers against homologous and heterologous tier 1 SIVs. Following 5 weekly intrarectal SIVmac251 challenges, two of sevenDNA/MVAand VLP (DM+VLP)-vaccinated animals were completely protected compared to productive infection in all seven DM-vaccinated animals. Vaccinated animals demonstrated stronger acute viral pulldown than controls, but a trend for higher acute viremia was observed in the DM+VLP group, likely due to a slower recall of Gag-specific CD8 T cells. Our findings support immunization with VLPs containing trimeric Env as a strategy to augment protective antibody but underscore the need for optimal engagement of CD8 T cells to achieve robust early viral control.
Background: In this study, we analyzed the protective efficacy of a simian immunodeficiency virus (SIV) macaque 239 (SIVmac239) analogue of the clinically tested GOVX-B11 deoxyribonucleic acid (DNA)/modified vaccinia Ankara (MVA) human immunodeficiency virus vaccine.
Methods: The tested vaccine used a DNA immunogen mutated to mimic the human vaccine and a regimen with DNA deliveries at weeks 0 and 8 and MVA deliveries at weeks 16 and 32. Twelve weekly rectal challenges with 0.3 animal infectious doses of SIV sootey mangabey E660 (SIVsmE660) were administered starting at 6 months after the last immunization.
Results: Over the first 6 rectal exposures to SIVsmE660, <10-year-old tripartite motif-containing protein 5 (TRIM5)α-permissive rhesus macaques showed an 80% reduction in per-exposure risk of infection as opposed to a 46% reduction in animals over 10 years old; and, over the 12 challenges, they showed a 72% as opposed to a 10% reduction. Analyses of elicited immune responses suggested that higher antibody responses in the younger animals had played a role in protection.
Conclusions: The simian analogue of the GOVX-B11 HIV provided strong protection against repeated rectal challenges in young adult macaques.
Background
Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines.
Principal Findings
We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg− recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2-fold) immunizations as compared to the udg+ control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVAΔudg-gag and MVA-gag. Furthermore, both udg− and udg+ MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 104–108 PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses.
Significance
Our identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the basis for a genetic system for MVA. DF-1 cells (and derivatives thereof) constitute viable alternatives, for the manufacture of MVA-based vaccines, to primary CEFs – the conventional cell substrate for MVA vaccines that is not amenable to genetic complementation strategies due to these cells' finite lifespan in culture. The establishment of a genetic system for MVA, as illustrated here to allow udg deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors.
UNLABELLED: It remains a challenge to develop a successful human immunodeficiency virus (HIV) vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a costimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We coexpressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia virus Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection, and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing antiviral humoral and cellular immunity, leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases, including HIV-1.
SARS-CoV-2 vaccines should induce broadly cross-reactive humoral and T cell responses to protect against emerging variants of concern (VOCs). Here, we inactivated the furin cleavage site (FCS) of spike expressed by a modified vaccinia Ankara (MVA) virus vaccine (MVA/SdFCS) and found that FCS inactivation markedly increased spike binding to human ACE2. After vaccination of mice, the MVA/SdFCS vaccine induced eightfold higher neutralizing antibodies compared with MVA/S, which expressed spike without FCS inactivation, and protected against the Beta variant. We next added nucleocapsid to the MVA/SdFCS vaccine (MVA/SdFCS-N) and tested its immunogenicity and efficacy via intramuscular (IM), buccal (BU), or sublingual (SL) routes in rhesus macaques. IM vaccination induced spike-specific IgG in serum and mucosae (nose, throat, lung, and rectum) that neutralized the homologous (WA-1/2020) and heterologous VOCs, including Delta, with minimal loss (<2-fold) of activity. IM vaccination also induced both spike- and nucleocapsid-specific CD4 and CD8 T cell responses in the blood. In contrast, the SL and BU vaccinations induced less spike-specific IgG in secretions and lower levels of polyfunctional IgG in serum compared with IM vaccination. After challenge with the SARS-CoV-2 Delta variant, the IM route induced robust protection, the BU route induced moderate protection, and the SL route induced no protection. Vaccine-induced neutralizing and non-neutralizing antibody effector functions positively correlated with protection, but only the effector functions correlated with early protection. Thus, IM vaccination with MVA/SdFCS-N vaccine elicited cross-reactive antibody and T cell responses, protecting against heterologous SARS-CoV-2 VOC more effectively than other routes of vaccination.
Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664. T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines.
Modified vaccinia Ankara (MVA) vector-based vaccines are attractive because of their excellent safety and ability to induce long-lived humoral and cellular immunity in humans. Routhu et al. show that an MVA-based COVID-19 vaccine encoding prefusion-stabilized spike (MVA/S) induces strong neutralizing antibody and CD8+ T cell responses and protects macaques from SARS-CoV2 infection, immunopathology, and infection-induced B cell abnormalities in the lungs.
Activated CD4 T cells are a major target of HIV infection. Results from the STEP HIV vaccine trial highlighted a potential role for total activated CD4 T cells in promoting HIV acquisition. However, the influence of vaccine insert-specific CD4 T cell responses on HIV acquisition is not known. Here, using the data obtained from four macaque studies, we show that the DNA prime/modified vaccinia Ankara boost vaccine induced interferon γ (IFNγ+) CD4 T cells [T helper 1 (TH1) cells] rapidly migrate to multiple tissues including colon, cervix, and vaginal mucosa. These mucosal TH1 cells persisted at higher frequencies and expressed higher density of CCR5, a viral coreceptor, compared to cells in blood. After intravaginal or intrarectal simian immunodeficiency virus (SIV)/simian-human immunodeficiency virus (SHIV) challenges, strong vaccine protection was evident only in animals that had lower frequencies of vaccine-specific TH1 cells but not in animals that had higher frequencies of TH1 cells, despite comparable vaccine-induced humoral and CD8 T cell immunity in both groups. An RNA transcriptome signature in blood at 7 days after priming immunization from one study was associated with induction of fewer TH1-type CD4 cells and enhanced protection. These results demonstrate that high and persisting frequencies of HIV vaccine–induced TH1-biased CD4 T cells in the intestinal and genital mucosa can mitigate beneficial effects of protective antibodies and CD8 T cells, highlighting a critical role of priming immunization and vaccine adjuvants in modulating HIV vaccine efficacy.
The RV144 human immunodeficiency virus type 1 (HIV-1) vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hot spot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA/simian-human immunodeficiency virus (SHIV) and boosting with modified vaccinia virus Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile.
Two gp140 booster immunizations induced very high levels (~2 mg/ml) of gp140 binding antibodies in serum, with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3, and gp41 immunodominant (ID) proteins. However, the vaccine-induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein-boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/ MVA vaccination do not significantly boost heterologous V1V2 and V2 HS responses and fail to enhance protection against heterologous SHIV challenge. IMPORTANCE HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually.
Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already wellestablished DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune cor- relates of protection in a moderately successful RV144 HIV vaccine trial in humans and highlight the need for the development of improved HIV envelope immunogens.
Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.