Passive Transfer of Vaccine-Elicited Antibodies Protects against SIV in Rhesus Macaques.

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.

Discovery and Selection of Hepatitis B Virus-Derived T Cell Epitopes for Global Immunotherapy Based on Viral Indispensability, Conservation, and HLA-Binding Strength.

Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.

Effective Suppression of HIV-1 Replication by Cytotoxic T Lymphocytes Specific for Pol Epitopes in Conserved Mosaic Vaccine Immunogens.

Cytotoxic T lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize circulating HIV-1 could be key for both HIV-1 cure and prophylaxis. We recently designed conserved mosaic T-cell vaccine immunogens (tHIVconsvX) composed of 6 Gag and Pol regions. Since the tHIVconsvX vaccine targets conserved regions common to most global HIV-1 variants and employs a bivalent mosaic design, it is expected that it could be universal if the vaccine works. Although we recently demonstrated that CTLs specific for 5 Gag epitopes in the vaccine immunogens had strong ability to suppress HIV-1 replication in vitro and in vivo, it remains unknown whether the Pol region-specific CTLs are equally efficient. In this study, we investigated CTLs specific for Pol epitopes in the immunogens in treatment-naive Japanese patients infected with HIV-1 clade B. Overall, we mapped 20 reported and 5 novel Pol conserved epitopes in tHIVconsvX. Responses to 6 Pol epitopes were significantly associated with good clinical outcome, suggesting that CTLs specific for these 6 Pol epitopes had a strong ability to suppress HIV-1 replication in HIV-1-infected individuals. In vitro T-cell analyses further confirmed that the Pol-specific CTLs could effectively suppress HIV-1 replication. The present study thus demonstrated that the Pol regions of the vaccine contained protective epitopes. T-cell responses to the previous 5 Gag and present 6 Pol protective epitopes together also showed a strong correlation with better clinical outcome. These findings support the testing of the conserved mosaic vaccine in HIV-1 cure and prevention in humans.IMPORTANCE It is likely necessary for an effective AIDS vaccine to elicit CD8+ T cells with the ability to recognize circulating HIV-1 and suppress its replication. We recently developed novel bivalent mosaic T-cell vaccine immunogens composed of conserved regions of the Gag and Pol proteins matched to at least 80% globally circulating HIV-1 isolates. Nevertheless, it remains to be proven if vaccination with these immunogens can elicit T cells with the ability to suppress HIV-1 replication. It is well known that Gag-specific T cells can suppress HIV-1 replication more effectively than T cells specific for epitopes in other proteins. We recently identified 5 protective Gag epitopes in the vaccine immunogens. In this study, we identified T cells specific for 6 Pol epitopes present in the immunogens with strong abilities to suppress HIV-1 in vivo and in vitro This study further encourages clinical testing of the conserved mosaic T-cell vaccine in HIV-1 prevention and cure.

CD8+ Cytotoxic T Lymphocyte Responses and Viral Epitope Escape in Acute HIV-1 Infection.

Epitope escape from HIV-1-targeted CD8+ cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8+ T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8+ T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8+ T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8+ CTL response pressure.

CD4+ T Cells Are Not Required for Suppression of Hepatitis B Virus Replication in the Liver of Vaccinated Chimpanzees.

Humans vaccinated with hepatitis B virus (HBV) surface antigen (HBsAg) sometimes develop humoral and cellular immunity to HBV proteins such as core and polymerase that are not vaccine components, providing indirect evidence that vaccine-induced immunity is not sterilizing. We previously described CD4(+) T-cell immunity against HBsAg and polymerase in chimpanzees after vaccination and HBV challenge. Here, vaccinated chimpanzees with protective levels of anti-HBsAg antibodies were rechallenged with HBV after antibody-mediated CD4(+) T-cell depletion. HBV DNA was detected in liver for at least 3 months after rechallenge, but virus replication was suppressed, as revealed by the absence of HBV DNA and HBsAg in serum. These observations provide direct virological evidence for nonsterilizing immunity in individuals with anti-HBsAg antibodies and are consistent with translation of HBV proteins to prime immune responses. They also indicate that CD4(+) T cells were not required for suppression of HBV replication in previously vaccinated individuals.

Protective efficacy of adenovirus/protein vaccines against SIV challenges in rhesus monkeys.

Preclinical studies of viral vector-based HIV-1 vaccine candidates have previously shown partial protection against neutralization-resistant virus challenges in rhesus monkeys. In this study, we evaluated the protective efficacy of adenovirus serotype 26 (Ad26) vector priming followed by purified envelope (Env) glycoprotein boosting. Rhesus monkeys primed with Ad26 vectors expressing SIVsmE543 Env, Gag, and Pol and boosted with AS01B-adjuvanted SIVmac32H Env gp140 demonstrated complete protection in 50% of vaccinated animals against a series of repeated, heterologous, intrarectal SIVmac251 challenges that infected all controls. Protective efficacy correlated with the functionality of Env-specific antibody responses. Comparable protection was also observed with a similar Ad/Env vaccine against repeated, heterologous, intrarectal SHIV-SF162P3 challenges. These data demonstrate robust protection by Ad/Env vaccines against acquisition of neutralization-resistant virus challenges in rhesus monkeys.

Comparative analysis of immunization schedules using a novel adenovirus-based immunotherapeutic targeting hepatitis B in naïve and tolerant mouse models.

Development of active targeted immunotherapeutics is a rapid developing field in the arena of chronic infectious diseases. The question of repeated, closely spaced administration of immunotherapeutics to achieve a rapid impact on the replicating agent is an important one. We analyzed here, using a prototype adenovirus-based immunotherapeutic encoding Core and Polymerase from the hepatitis B virus (Ad-HBV), the influence of closely spaced repeated immunizations on the level and quality of induced HBV-specific and vector-specific immune responses in various mouse models. Ad-HBV, whether injected once or multiple times, was able to induce HBV- and adeno-specific T cells both in HBV-free mice and in a HBV tolerant mouse model. Adenovirus-specific T cell responses and titers of neutralizing anti-Ad5 antibodies increased from time of the 3rd injection. Interestingly, single or multiple Ad-HBV injections resulted in detection of Polymerase-specific functional T cells in HBV tolerant mice. Overall no modulation of the levels of HBV-specific cytokine-producing (IFNγ/TNFα) and cytolytic T cells was observed following repeated administrations (3 or 6 weekly injections) when compared with levels detected after a single injection with the exception of two markers: 1. the proportion of HBV-specific IFNγ-producing cells bearing the CD27+/CD43+ phenotype appeared to be sustained in C57BL/6J mice following 6 weekly injections; 2. the percentage of IFNγ/TNFα Core-specific producing cells observed in spleens of HLA-A2 mice as well as of that specific of Polymerase observed in livers of HBV tolerant mice was maintained. In addition, percentage of HBV-specific T cells expressing PD-1 was not increased by multiple injections. Overall these data show that, under experimental conditions used, rapid, closely spaced administrations of an adenovirus-based HBV immunotherapeutics does not inhibit induced T-cell responses including in a HBV-tolerant environment.

GagPol-specific CD4⁺ T-cells increase the antibody response to Env by intrastructural help.

BACKGROUND: Immunization of rhesus macaques against Gag of SIV resulted in a more rapid appearance of Env antibodies after infection with SIV or SHIV challenge viruses although the vaccines lacked an Env component. We therefore explored whether T helper cells specific for internal HIV proteins could provide intrastructural help for Env-specific B cells and thus increase the Env antibody response. RESULTS: Mice were immunized by adenoviral vector or DNA vaccines against GagPol and then boosted with virus-like particles (VLP) containing GagPol and Env. Env-specific antibody levels after the VLP booster immunizations were significantly higher in GagPol-immunized mice than in mock-vaccinated controls. Adoptive transfer of CD4+ T cells from GagPol-immunized mice also enhanced the Env antibody response to VLP immunization in the recipient mice. Depending on the presence of VLPs, co-cultivation of CD4+ T cells from GagPol-primed mice with BCR transgenic B cells specific for a protein presented on the surface of the VLPs also resulted in the activation of the B and T cells. CONCLUSIONS: Our study indicates that GagPol-specific T helper cells may provide intrastructural help for Env antibody responses. This cross-talk between immune responses directed against different components of the retroviral particle may be relevant for the immunopathogenesis of retroviral infections and allow to improve virus like particle vaccine approaches against HIV.

Mucosal priming with a replicating-vaccinia virus-based vaccine elicits protective immunity to simian immunodeficiency virus challenge in rhesus monkeys.

Mucosal surfaces are not targeted by most human immunodeficiency virus type 1 (HIV-1) vaccines, despite being major routes for HIV-1 transmission. Here we report a novel vaccination regimen consisting of a mucosal prime with a modified replicating vaccinia virus Tiantan strain (MVTT(SIVgpe)) and an intramuscular boost with a nonreplicating adenovirus strain (Ad5(SIVgpe)). This regimen elicited robust cellular immune responses with enhanced magnitudes, sustainability, and polyfunctionality, as well as higher titers of neutralizing antibodies against the simian immunodeficiency virus SIV(mac1A11) in rhesus monkeys. The reductions in peak and set-point viral loads were significant in most animals, with one other animal being protected fully from high-dose intrarectal inoculation of SIV(mac239). Furthermore, the animals vaccinated with this regimen were healthy, while ~75% of control animals developed simian AIDS. The protective effects correlated with the vaccine-elicited SIV-specific CD8(+) T cell responses against Gag and Pol. Our study provides a novel strategy for developing an HIV-1 vaccine by using the combination of a replicating vector and mucosal priming.

Frequent detection of herpes simplex virus antigen in skin and peripheral blood CD34+ mononuclear cells from patients with graft-versus-host disease.

Viruses are implicated in the initiation or flare of graft-versus-host disease (GVHD) by virtue of their ability to activate antigen-presenting dendritic cells (DC). Herpes simplex virus (HSV) infects circulating CD34+ stem cell progenitors, favoring their differentiation into skin homing DC (CD1a+ Langerhans cells) that contribute to the development of an inflammatory skin rash known as HSV-associated erythema multiforme (HAEM). Following on these findings, we conducted a prospective study to examine whether HSV is also associated with GVHD. Skin biopsies and peripheral blood mononuclear cells (PBMC) were collected from 37 consecutive patients on admission before and after allogeneic hematopoietic stem cell transplantation (HSCT) and examined for HSV antigen (Pol) expression and the presence of Pol+CD34+ and Pol+CD1a+ cells. Sixteen patients developed a skin rash that was histopathologically consistent with GVHD (group I), 3 patients had a rash that was not GVHD (group II, EM-like) and 18 patients did not develop any rash after HSCT (group III). Skin biopsies from the group I patients were Pol negative pre-HSCT (baseline) but became Pol+ after the diagnosis of GVHD. The GVHD biopsies also contained Pol+CD34+ and Pol+CD1a+ cells, and these patients had a significant percentage of circulating Pol+CD34+ and Pol+CD1a+ PBMC. By contrast, the group II patients had Pol+ skin cells and Pol+CD34+ circulating PBMC at baseline that decreased post-HSCT. The group III patients had Pol negative skin and very few circulating Pol+CD34+ and Pol+CD1a+ PBMC at baseline that were not significantly changed post-HSCT. The data associate skin GVHD with HSV reactivation during conditioning and its propensity for nonreplicative infection of CD34+ PBMC that induces DC activation. Further studies are needed to better elucidate this association.

Circumventing antivector immunity by using adenovirus-infected blood cells for repeated application of adenovirus-vectored vaccines: proof of concept in rhesus macaques.

Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14(+) cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.

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.

Inclusion of a CRF01_AE HIV envelope protein boost with a DNA/MVA prime-boost vaccine: Impact on humoral and cellular immunogenicity and viral load reduction after SHIV-E challenge.

The current study assessed the immunogenicity and protective efficacy of various prime-boost vaccine regimens in rhesus macaques using combinations of recombinant DNA (rDNA), recombinant MVA (rMVA), and subunit gp140 protein. The rDNA and rMVA vectors were constructed to express Env from HIV-1 subtype CRF01_AE and Gag-Pol from CRF01_AE or SIVmac 239. One of the rMVAs, MVA/CMDR, has been recently tested in humans. Immunizations were administered at months 0 and 1 (prime) and months 3 and 6 (boost). After priming, HIV env-specific serum IgG was detected in monkeys receiving gp140 alone or rMVA but not in those receiving rDNA. Titers were enhanced in these groups after boosting either with gp140 alone or with rMVA plus gp140. The groups that received the rDNA prime developed env-specific IgG after boosting with rMVA with or without gp140. HIV Env-specific serum IgG binding antibodies were elicited more frequently and of higher titer, and breadth of neutralizing antibodies was increased with the inclusion of the subunit Env boost. T cell responses were measured by tetramer binding to Gag p11c in Mamu-A*01 macaques, and by IFN-γ ELISPOT assay to SIV-Gag. T cell responses were induced after vaccination with the highest responses seen in macaques immunized with rDNA and rMVA. Macaques were challenged intravenously with a novel SHIV-E virus (SIVmac239 Gag-Pol with an HIV-1 subtype E-Env CAR402). Post challenge with SHIV-E, antibody titers were boosted in all groups and peaked at 4 weeks. Robust T cell responses were seen in all groups post challenge and in macaques immunized with rDNA and rMVA a clear boosting of responses was seen. A greater than two-log drop in RNA copies/ml at peak viremia and earlier set point was achieved in macaques primed with rDNA, and boosted with rMVA/SHIV-AE plus gp140. Post challenge viremia in macaques immunized with other regimens was not significantly different to that of controls. These results demonstrate that a gp140 subunit and inclusion of SIV Gag-Pol may be critical for control of SHIV post challenge.

Coexistence of hepatitis B surface antigen and anti-HBs in Chinese chronic hepatitis B virus patients relating to genotype C and mutations in the S and P gene reverse transcriptase region.

We aimed to determine the prevalence of the coexistence of HBsAg and anti-HBs and to analyze the clinical and virological features of infection, including amino acid (aa) patterns of the S gene and reverse transcriptase (RT) region in Chinese chronic hepatitis B (CHB) patients. Fifty-four (2.90%) CHB patients who were positive for both HBsAg and anti-HBs were tested, and sequences were obtained from 52 of them as well as 48 patients from a control group. S gene and RT region sequences were amplified and sequenced using in-house protocols. There was no significant difference between patients with and without anti-HBs with regard to age, gender, alanine aminotransferase level, and the proportion positive for HBeAg and HBcAb. The occurrence of genotype C (P = 0.001) and anti-HBeAb positivity (P = 0.027) was significantly higher in HBsAg+/anti-HBs+ individuals. In the S gene, the number of mutated residues in the HBsAg+/anti-HBs+ group was markedly higher than in control patients (1.88 versus 1.02 substitutions per 100 amino acids, P = 0.022). The amino acid exchange occurred mostly within the N-terminal region (2.15 versus 0.87 substitutions per 100 amino acids, P = 0.023) and the "a" determinant (3.61 versus 1.56 substitutions per 100 amino acids, P = 0.049) in the two groups. In the RT region, the mean number of substitution per 100 aa showed a tendency to be significantly higher in HBsAg+/anti-HBs+ patients than in controls (2.34 versus 1.46, P = 0.040). This study showed a prevalence of coexistence of anti-HBs in HBsAg-positive patients and an increased frequency of genotype C and aa variability within both HBsAg and RT involving functionally important regions of those proteins.

HIV-DNA priming alters T cell responses to HIV-adenovirus vaccine even when responses to DNA are undetectable.

Many candidate HIV vaccines are designed to primarily elicit T cell responses. Although repeated immunization with the same vaccine boosts Ab responses, the benefit for T cell responses is ill defined. We compared two immunization regimens that include the same recombinant adenoviral serotype 5 (rAd5) boost. Repeated homologous rAd5 immunization fails to increase T cell responses, but increases gp140 Ab responses 10-fold. DNA prime, as compared with rAd5 prime, directs long-term memory CD8(+) T cells toward a terminally differentiated effector memory phenotype with cytotoxic potential. Based on the kinetics of activated cells measured directly ex vivo, the DNA vaccination primes for both CD4(+) and CD8(+) T cells, despite the lack of detection of the latter until after the boost. These results suggest that heterologous prime-boost combinations have distinct immunological advantages over homologous prime-boosts and suggest that the effect of DNA on subsequent boosting may not be easily detectable directly after the DNA vaccination.

Induction and comparison of SIV immunity in Ad5 naïve and Ad5 immune non-human primates using an Ad5 [E1-, E2b-] based vaccine.

The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.

PAComplex: a web server to infer peptide antigen families and binding models from TCR-pMHC complexes.

One of the most adaptive immune responses is triggered by specific T-cell receptors (TCR) binding to peptide-major histocompatibility complexes (pMHC). Despite the availability of many prediction servers to identify peptides binding to MHC, these servers are often lacking in peptide-TCR interactions and detailed atomic interacting models. PAComplex is the first web server investigating both pMHC and peptide-TCR interfaces to infer peptide antigens and homologous peptide antigens of a query. This server first identifies significantly similar TCR-pMHC templates (joint Z-value ≥ 4.0) of the query by using antibody-antigen and protein-protein interacting scoring matrices for peptide-TCR and pMHC interfaces, respectively. PAComplex then identifies the homologous peptide antigens of these hit templates from complete pathogen genome databases (≥10(8) peptide candidates from 864,628 protein sequences of 389 pathogens) and experimental peptide databases (80,057 peptides in 2287 species). Finally, the server outputs peptide antigens and homologous peptide antigens of the query and displays detailed interacting models (e.g. hydrogen bonds and steric interactions in two interfaces) of hitTCR-pMHC templates. Experimental results demonstrate that the proposed server can achieve high prediction accuracy and offer potential peptide antigens across pathogens. We believe that the server is able to provide valuable insights for the peptide vaccine and MHC restriction. The PAComplex sever is available at http://PAcomplex.life.nctu.edu.tw.

Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys.

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.

Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion.

Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IkappaB kinase-epsilon (IKKepsilon), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKepsilon activity by disrupting the interaction between IKKepsilon and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKepsilon activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.

Vaccine-induced IgG2 anti-HIV p24 is associated with control of HIV in patients with a 'high-affinity' FcgammaRIIa genotype.

OBJECTIVES: We have previously shown that vaccination with a recombinant fowlpox virus carrying the genes for HIV Gag-Pol and interferon-gamma (IFN-gamma) was associated with partial control of HIV replication after antiretroviral therapy (ART) was ceased but not with increased anti-HIV T-cell responses. Because IFN-gamma enhances IgG2 production, and IgG2 antibodies to HIV antigens and the 'high-affinity' polymorphism of FcgammaRIIa (the major Fc receptor for IgG2) have been associated with a favourable outcome of HIV infection, we examined the association of IgG2 antibodies to HIV p24 and 'high-affinity' polymorphisms of FcgammaRIIa with control of HIV replication in these patients. METHODS: Plasma from weeks 0 (cessation of ART 1 week after the last vaccination), 9 and 20 was available from patients who had received the full construct vaccine, a partial construct (without IFN-gamma) or placebo. IgG2 and IgG1 anti-p24 and anti-gp41 were assayed and all patients were genotyped for the FcgammaRIIa 131 R/H polymorphism that affects IgG2 binding. RESULTS: At week 0, IgG2 anti-p24 was present in five of nine full construct patients but none of 14 partial construct or placebo patients and was associated with a smaller increase in plasma HIV RNA over 20 weeks. Patients with IgG2 anti-p24 and the 'high-affinity' polymorphism of FcgammaRIIa exhibited lower HIV replication than other patients at week 20. CONCLUSION: The role of IgG2 anti-HIV antibodies and FcgammaRIIa in the control of HIV replication should be investigated further. Inclusion of an IFN-gamma gene in DNA vaccine constructs might be a means of enhancing IgG2 antibody production.