Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.

Evaluation of heterologous prime-boost vaccination strategies using chimpanzee adenovirus and modified vaccinia virus for TB subunit vaccination in rhesus macaques.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

Prevention of tuberculosis in macaques after intravenous BCG immunization.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide1. The only available vaccine, BCG (Bacillus Calmette-Guérin), is given intradermally and has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission1,2. Here we show that intravenous administration of BCG profoundly alters the protective outcome of Mtb challenge in non-human primates (Macaca mulatta). Compared with intradermal or aerosol delivery, intravenous immunization induced substantially more antigen-responsive CD4 and CD8 T cell responses in blood, spleen, bronchoalveolar lavage and lung lymph nodes. Moreover, intravenous immunization induced a high frequency of antigen-responsive T cells across all lung parenchymal tissues. Six months after BCG vaccination, macaques were challenged with virulent Mtb. Notably, nine out of ten macaques that received intravenous BCG vaccination were highly protected, with six macaques showing no detectable levels of infection, as determined by positron emission tomography-computed tomography imaging, mycobacterial growth, pathology and granuloma formation. The finding that intravenous BCG prevents or substantially limits Mtb infection in highly susceptible rhesus macaques has important implications for vaccine delivery and clinical development, and provides a model for defining immune correlates and mechanisms of vaccine-elicited protection against tuberculosis.

A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

Immune response and reactogenicity of an unadjuvanted intradermally delivered human papillomavirus vaccine using a first generation Nanopatch™ in rhesus macaques: An exploratory, pre-clinical feasibility assessment.

The human papillomavirus (HPV) 9-valent, recombinant vaccine (Gardasil™9) helps protect young adults (males and females) against anogenital cancers and genital warts caused by certain HPV genotypes (ref. Gardasil™9 insert). This vaccine is administered intramuscularly (IM). The aim of this study was to determine preclinically whether intradermal (ID) vaccination with an unadjuvanted 9-valent recombinant HPV vaccine using a first-generation ID delivery device, the Nanopatch™, could enhance vaccine immunogenicity compared with the traditional ID route (Mantoux technique). IM injection of HPV VLPs formulated with Merck & Co., Inc., Kenilworth, NJ, USA Alum Adjuvant (MAA) were included in the rhesus study for comparison. The Nanopatch™ prototype contains a high-density array comprised of 10,000 microprojections/cm2, each 250 µm long. It was hypothesized the higher density array with shallower ID delivery may be superior to the Mantoux technique. To test this hypothesis, HPV VLPs without adjuvant were coated on the Nanopatch™, stability of the Nanopatch™ with unadjuvanted HPV VLPs were evaluated under accelerated conditions, skin delivery was verified using radiolabelled VLPs or FluoSpheres®, and the immune response and skin site reaction with the Nanopatch™ was evaluated in rhesus macaques. The immune response induced by Nanopatch™ administration, measured as HPV-specific binding antibodies, was similar to that induced using the Mantoux technique. It was also observed that a lower dose of unadjuvanted HPV VLPs delivered with the first-generation Nanopatch™ and applicator or Mantoux technique resulted in an immune response that was significantly lower compared to a higher-dose of alum adjuvanted HPV VLPs delivered IM in rhesus macaques. The study also indicated unadjuvanted HPV VLPs could be delivered with the first-generation Nanopatch™ and applicator to the skin in 15 s with a transfer efficiency of approximately 20%. This study is the first demonstration of patch administration in non-human primates with a vaccine composed of HPV VLPs.

Functionally inactivated dominant viral antigens of human cytomegalovirus delivered in replication incompetent adenovirus type 6 vectors as vaccine candidates.

T cell immunity is critical in controlling human cytomegalovirus (HCMV) infection in transplant recipients, and T cells targeting viral immediate early proteins such as IE1, IE2 and pp65 have been speculated to be more effective against reactivation. Here we report efforts to construct replication incompetent adenovirus 6 vectors expressing these viral antigens as vaccine candidates. To reduce the potential liabilities of these viral proteins as vaccine antigens, we introduced mutations to inactivate their reported functions including their nuclear localization signals. The modifications greatly reduced their localization to the nuclei, thus limiting their interactions with cellular proteins important for cell cycle modulation and transactivation. The immunogenicity of modified pp65, IE1 and IE2 vaccines was comparable to their wild-type counterparts in mice and the immunogenicity of the modified antigens was demonstrated in non-human primates.

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates.

Dengue virus has emerged as an important arboviral infection worldwide. As a complex pathogen, with four distinct serotypes, the development of a successful Dengue virus vaccine has proven to be challenging. Here, we describe a novel Dengue vaccine candidate that contains truncated, recombinant, Dengue virus envelope protein from all four Dengue virus serotypes (DEN-80E) formulated with ionizable cationic lipid nanoparticles (LNPs). Immunization studies in mice, Guinea pigs, and in Rhesus macaques, revealed that LNPs induced high titers of Dengue virus neutralizing antibodies, with or without co-administration or encapsulation of a Toll-Like Receptor 9 agonist. Importantly, LNPs were also able to boost DEN-80E specific CD4+ and CD8+ T cell responses. Cytokine and chemokine profiling revealed that LNPs induced strong chemokine responses without significant induction of inflammatory cytokines. In addition to being highly efficacious, the vaccine formulation proved to be well-tolerated, demonstrating no elevation in any of the safety parameters evaluated. Notably, reduction in cationic lipid content of the nanoparticle dramatically reduced the LNP's ability to boost DEN-80E specific immune responses, highlighting the crucial role for the charge of the LNP. Overall, our novel studies, across multiple species, reveal a promising tetravalent Dengue virus sub-unit vaccine candidate.

Characterization of N-glycosylation profiles from mammalian and insect cell derived chikungunya VLP.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe arthralgia. The envelope of CHIKV is composed of 240 copies of two glycoproteins: E1 and E2. In this work, we have characterized the N-glycosylation patterns of CHIKV virus-like particles (VLPs), containing both E1 and E2 proteins, derived from mammalian and insect cells using hydrophilic interaction liquid chromatography (HILIC) with fluorescence (FL) and mass spectrometry (MS) detection. While HEK293 derived CHIKV VLPs contain oligomannose, hybrid and complex glycans, VLPs derived from SfBasic predominantly contain oligomannose glycans. This strong host dependence of N-glycosylation pattern resembles other alphaviruses such as SINV. The VLPs from HEK293 and SfBasic, with significantly different N-glycosylation profiles, are valuable reagents enabling future in-depth correlation studies between immunogenicity and glycosylation. In addition, the characterization tools presented here allow one to monitor glycosylation during vaccine process development and ensure process consistency.

Rapid isolation of dengue-neutralizing antibodies from single cell-sorted human antigen-specific memory B-cell cultures.

Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen-specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here, we describe a flow cytometry method to identify and isolate dengue envelope-specific memory B cells using a labeled dengue envelope protein. We enumerated dengue-envelope specific memory B cells from a cohort of dengue seropositive donors using this direct flow cytometry assay. A more established and conventional assay, the cultured B ELISPOT, was used as a benchmark comparator. Furthermore, we were able to confirm the single-sorted memory B-cell specificity by culturing B cells and differentiating them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them as full-length recombinant antibodies to reproduce the activity seen in culture supernatants. Mapping of these antibodies revealed a novel epitope for dengue 2 virus serotype. In conclusion, we established a reproducible methodology to enumerate antigen-specific memory B cells and assay their encoded antibodies for functional characterization.

A herpes simplex virus 2 (HSV-2) glycoprotein D-expressing nonreplicating dominant-negative HSV-2 virus vaccine is superior to a gD2 subunit vaccine against HSV-2 genital infection in guinea pigs.

We recently constructed a novel non-replicating dominant-negative HSV-2 recombinant viral vaccine (CJ2-gD2) capable of expressing various HSV-2 antigens that are dominant targets of HSV-2-specific CD8 T-cell response. Importantly, CJ2-gD2 expresses gD2, the HSV-2 major antigen glycoprotein D, as efficiently as wild-type HSV-2 infection and can lead to a nearly 500-fold reduction in wild-type HSV-2 viral replication in cells co-infected with CJ2-gD2 and wild-type HSV-2. In this report, we show that CJ2-gD2 elicits a strong antibody response to various HSV-2 antigens and is highly effective in the prevention of primary and recurrent HSV-2 genital infection and disease in the immunized guinea pigs. The direct comparison study between CJ2-gD2 and a gD2 subunit vaccine (gD2-alum/MPL) with a formulation akin to a vaccine tested in phase III clinical trials shows that CJ2-gD2 is 8 times more effective than the gD2-alum/MPL subunit vaccine in eliciting an anti-HSV-2 specific neutralizing antibody response and offers significantly superior protection against primary and recurrent HSV-2 genital infections. Importantly, no challenge wild-type HSV-2 viral DNA was detectable in dorsal root ganglia DNA isolated from CJ2-gD2-immunized guinea pigs on day 60 post-challenge. CJ2-gD2 should be an excellent HSV-2 vaccine candidate for protection against HSV-2 genital infection and disease in humans.

Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line.

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2-6.4), properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6-6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0-7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.

Protection provided by a herpes simplex virus 2 (HSV-2) glycoprotein C and D subunit antigen vaccine against genital HSV-2 infection in HSV-1-seropositive guinea pigs.

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.

Pentameric complex of viral glycoprotein H is the primary target for potent neutralization by a human cytomegalovirus vaccine.

Human cytomegalovirus (HCMV) can cause serious morbidity/mortality in transplant patients, and congenital HCMV infection can lead to birth defects. Developing an effective HCMV vaccine is a high medical priority. One of the challenges to the efforts has been our limited understanding of the viral antigens important for protective antibodies. Receptor-mediated viral entry to endothelial/epithelial cells requires a glycoprotein H (gH) complex comprising five viral proteins (gH, gL, UL128, UL130, and UL131). This gH complex is notably missing from HCMV laboratory strains as well as HCMV vaccines previously evaluated in the clinic. To support a unique vaccine concept based on the pentameric gH complex, we established a panel of 45 monoclonal antibodies (mAbs) from a rabbit immunized with an experimental vaccine virus in which the expression of the pentameric gH complex was restored. Over one-half (25 of 45) of the mAbs have neutralizing activity. Interestingly, affinity for an antibody to bind virions was not correlated with its ability to neutralize the virus. Genetic analysis of the 45 mAbs based on their heavy- and light-chain sequences identified at least 26 B-cell linage groups characterized by distinct binding or neutralizing properties. Moreover, neutralizing antibodies possessed longer complementarity-determining region 3 for both heavy and light chains than those with no neutralizing activity. Importantly, potent neutralizing mAbs reacted to the pentameric gH complex but not to gB. Thus, the pentameric gH complex is the primary target for antiviral antibodies by vaccination.

Immunogenicity of a reduced-dose whole killed rabies vaccine is significantly enhanced by ISCOMATRIX™ adjuvant, Merck amorphous aluminum hydroxylphosphate sulfate (MAA) or a synthetic TLR9 agonist in rhesus macaques.

There is a need for novel rabies vaccines suitable for short course, pre- and post-exposure prophylactic regimens which require reduced doses of antigen to address the current worldwide supply issue. We evaluated in rhesus macaques the immunogenicity of a quarter-dose of a standard rabies vaccine formulated with Merck's amorphous aluminum hydroxylphosphate sulfate adjuvant, the saponin-based ISCOMATRIX™ adjuvant, or a synthetic TLR9 agonist. All adjuvants significantly increased the magnitude and durability of the humoral immune response as measured by rapid fluorescent focus inhibition test (RFFIT). Several three-dose vaccine regimens resulted in adequate neutralizing antibody of ≥ 0.5 IU/ml earlier than the critical day seven post the first dose. Rabies vaccine with ISCOMATRIX™ adjuvant given at days 0 and 3 resulted in neutralizing antibody titers which developed faster and were up to one log10 higher compared to WHO-recommended intramuscular and intradermal regimens and furthermore, passive administration of human rabies immunoglobulin did not interfere with immunogenicity of this reduced dose, short course vaccine regimen. Adjuvantation of whole-killed rabies vaccine for intramuscular injection may therefore be a viable alternative to intradermal application of non-adjuvanted vaccine for both pre- and post-exposure regimens.

HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.

An efficient T-cell epitope discovery strategy using in silico prediction and the iTopia assay platform.

Functional T-cell epitope discovery is a key process for the development of novel immunotherapies, particularly for cancer immunology. In silico epitope prediction is a common strategy to try to achieve this objective. However, this approach suffers from a significant rate of false-negative results and epitope ranking lists that often are not validated by practical experience. A high-throughput platform for the identification and prioritization of potential T-cell epitopes is the iTopia(TM) Epitope Discovery System(TM), which allows measuring binding and stability of selected peptides to MHC Class I molecules. So far, the value of iTopia combined with in silico epitope prediction has not been investigated systematically. In this study, we have developed a novel in silico selection strategy based on three criteria: (1) predicted binding to one out of five common MHC Class I alleles; (2) uniqueness to the antigen of interest; and (3) increased likelihood of natural processing. We predicted in silico and characterized by iTopia 225 candidate T-cell epitopes and fixed-anchor analogs from three human tumor-associated antigens: CEA, HER2 and TERT. HLA-A2-restricted fragments were further screened for their ability to induce cell-mediated responses in HLA-A2 transgenic mice. The iTopia binding assay was only marginally informative while the stability assay proved to be a valuable experimental screening method complementary to in silico prediction. Thirteen novel T-cell epitopes and analogs were characterized and additional potential epitopes identified, providing the basis for novel anticancer immunotherapies. In conclusion, we show that combination of in silico prediction and an iTopia-based assay may be an accurate and efficient method for MHC Class I epitope discovery among tumor-associated antigens.

Restoration of viral epithelial tropism improves immunogenicity in rabbits and rhesus macaques for a whole virion vaccine of human cytomegalovirus.

Maternal immunity to human cytomegalovirus (HCMV) prior to conception is ~70% protective against congenital transmission and in utero infection of HCMV. Both functional antibodies capable of neutralizing virus and effective T-cells are believed to be important for the protection. Previous HCMV vaccines have rarely been shown able to induce neutralizing antibody titers comparable to those seen in naturally infected HCMV seropositive subjects. Recent studies link a glycoprotein H (gH) complex to receptor-mediated viral entry of endothelial/epithelial cells and leukocytes. This pentameric gH complex, composed of five proteins (gH, gL, UL128, UL130 and UL131 proteins), is notably missing in all HCMV vaccine previously evaluated in clinic. Here we showed that a HCMV virus, with restored expression of the pentameric gH complex, can induce 10-fold higher neutralizing antibody titers than an attenuated AD169 virus or a recombinant glycoprotein B vaccine in multiple animal species in which viral replication is not expected. Encouragingly, the peak neutralizing titers post vaccination in rabbits and monkeys were within 2-4-fold of the levels determined in HCMV seropositive subjects. Functional antibodies by vaccination could further be improved when formulated with a novel adjuvant, and the titers of the antiviral antibodies were sustained in rabbits for over a year after vaccination. These results indicate that the pentameric gH complex is associated with greatly improved functional antibodies following vaccination, and support a vaccine concept based on a nonreplicating whole HCMV with the pentameric gH-associated epithelial tropism restored.

A trivalent recombinant Ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous SIV challenge.

It has been suggested that poor immunogenicity may explain the lack of vaccine efficacy in preventing or controlling HIV infection in the Step trial. To investigate this issue we vaccinated eight Indian rhesus macaques with a trivalent replication-incompetent adenovirus serotype 5 vaccine expressing SIV Gag, Pol, and Nef using a regimen similar to that employed in the Step trial. We detected broad vaccine-induced CD8(+) (2-7 pool-specific responses) and CD4(+) (5-19 pool-specific responses) T-cell responses in IFN-γ ELISPOT assays at one week post-boost using fresh PBMC. However, using cryopreserved cells at one and four weeks post-boost we observed a reduction in both the number and magnitude of most vaccine-induced responses. This demonstrates that the time points and conditions chosen to perform immune assays may influence the observed breadth and frequency of vaccine-induced T-cell responses. To evaluate protective efficacy, we challenged the immunized macaques, along with naïve controls, with repeated, limiting doses of the heterologous swarm isolate SIVsmE660. Vaccination did not significantly affect acquisition or control of virus replication in vaccinees compared to naïve controls. Post-infection we observed an average of only two anamnestic CD8(+) T-cell responses per animal, which may not have been sufficiently broad to control heterologous virus replication. While the trivalent vaccine regimen induced relatively broad T-cell responses in rhesus macaques, it failed to protect against infection or control viral replication. Our results are consistent with those observed in the Step trial and indicate that SIV immunization and challenge studies in macaque models of HIV infection can be informative in assessing pre-clinical HIV vaccines.

The number of Toll-like receptor 9-agonist motifs in the adenovirus genome correlates with induction of dendritic cell maturation by adenovirus immune complexes.

Adenovirus serotype 5 (Ad5) vectors and specific neutralizing antibodies (NAbs) generate immune complexes (ICs) which are potent inducers of dendritic cell (DC) maturation. Here we show that ICs generated with rare Ad vector serotypes, such as Ad26 and Ad35, which are lead candidates in HIV vaccine development, are poor inducers of DC maturation and that their potency in inducing DC maturation strongly correlated with the number of Toll-like receptor 9 (TLR9)-agonist motifs present in the Ad vector's genome. In addition, we showed that antihexon but not antifiber antibodies are responsible for the induction of Ad IC-mediated DC maturation.

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread.

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.