Recombinant Herpesvirus Vectors: Durable Immune Responses and Durable Protection against Simian Immunodeficiency Virus SIVmac239 Acquisition.

A prophylactic vaccine that confers durable protection against human immunodeficiency virus (HIV) would provide a valuable tool to prevent new HIV/AIDS cases. As herpesviruses establish lifelong infections that remain largely subclinical, the use of persistent herpesvirus vectors to deliver HIV antigens may facilitate the induction of long-term anti-HIV immunity. We previously developed recombinant (r) forms of the gamma-herpesvirus rhesus monkey rhadinovirus (rRRV) expressing a replication-incompetent, near-full-length simian immunodeficiency virus (SIVnfl) genome. We recently showed that 8/16 rhesus macaques (RMs) vaccinated with a rDNA/rRRV-SIVnfl regimen were significantly protected against intrarectal (i.r.) challenge with SIVmac239. Here we investigated the longevity of this vaccine-mediated protection. Despite receiving no additional booster immunizations, the protected rDNA/rRRV-SIVnfl vaccinees maintained detectable cellular and humoral anti-SIV immune responses for more than 1.5 years after the rRRV boost. To assess if these responses were still protective, the rDNA/rRRV-SIVnfl vaccinees were subjected to a second round of marginal-dose i.r. SIVmac239 challenges, with eight SIV-naive RMs serving as concurrent controls. After three SIV exposures, 8/8 control animals became infected, compared to 3/8 vaccinees. This difference in SIV acquisition was statistically significant (P = 0.0035). The three vaccinated monkeys that became infected exhibited significantly lower viral loads than those in unvaccinated controls. Collectively, these data illustrate the ability of rDNA/rRRV-SIVnfl vaccination to provide long-term immunity against stringent mucosal challenges with SIVmac239. Future work is needed to identify the critical components of this vaccine-mediated protection and the extent to which it can tolerate sequence mismatches in the challenge virus. IMPORTANCE We report on the long-term follow-up of a group of rhesus macaques (RMs) that received an AIDS vaccine regimen and were subsequently protected against rectal acquisition of simian immunodeficiency virus (SIV) infection. The vaccination regimen employed included a live recombinant herpesvirus vector that establishes persistent infection in RMs. Consistent with the recurrent SIV antigen expression afforded by this herpesvirus vector, vaccinees maintained detectable SIV-specific immune responses for more than 1.5 years after the last vaccination. Importantly, these vaccinated RMs were significantly protected against a second round of rectal SIV exposures performed 1 year after the first SIV challenge phase. These results are relevant for HIV vaccine development because they show the potential of herpesvirus-based vectors to maintain functional antiretroviral immunity without the need for repeated boosting.

Vaccine protection against SIVmac239 acquisition.

The biological characteristics of HIV pose serious difficulties for the success of a preventive vaccine. Molecularly cloned SIVmac239 is difficult for antibodies to neutralize, and a variety of vaccine approaches have had great difficulty achieving protective immunity against it in rhesus monkey models. Here we report significant protection against i.v. acquisition of SIVmac239 using a long-lasting approach to vaccination. The vaccine regimen includes a replication-competent herpesvirus engineered to contain a near-full-length SIV genome that expresses all nine SIV gene products, assembles noninfectious SIV virion particles, and is capable of eliciting long-lasting effector-memory cellular immune responses to all nine SIV gene products. Vaccinated monkeys were significantly protected against acquisition of SIVmac239 following repeated marginal dose i.v. challenges over a 4-month period. Further work is needed to define the critical components necessary for eliciting this protective immunity, evaluate the breadth of the protection against a variety of strains, and explore how this approach may be extended to human use.

A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses.

A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naive rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real-time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ versus CD20+ versus CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro, in which deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks, Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag181-189CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors are dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role for gL and Eph family receptors in B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

Rectal Acquisition of Simian Immunodeficiency Virus (SIV) SIVmac239 Infection despite Vaccine-Induced Immune Responses against the Entire SIV Proteome.

Given the complex biology of human immunodeficiency virus (HIV) and its remarkable capacity to evade host immune responses, HIV vaccine efficacy may benefit from the induction of both humoral and cellular immune responses of maximal breadth, potency, and longevity. Guided by this rationale, we set out to develop an immunization protocol aimed at maximizing the induction of anti-Envelope (anti-Env) antibodies and CD8+ T cells targeting non-Env epitopes in rhesus macaques (RMs). Our approach was to deliver the entire simian immunodeficiency virus (SIV) proteome by serial vaccinations. To that end, 12 RMs were vaccinated over 81 weeks with DNA, modified vaccinia Ankara (MVA), vesicular stomatitis virus (VSV), adenovirus type 5 (Ad5), rhesus monkey rhadinovirus (RRV), and DNA again. Both the RRV and the final DNA boosters delivered a near-full-length SIVmac239 genome capable of assembling noninfectious SIV particles and inducing T-cell responses against all nine SIV proteins. Compared to previous SIV vaccine trials, the present DNA-MVA-VSV-Ad5-RRV-DNA regimen resulted in comparable levels of Env-binding antibodies and SIV-specific CD8+ T-cells. Interestingly, one vaccinee developed low titers of neutralizing antibodies (NAbs) against SIVmac239, a tier 3 virus. Following repeated intrarectal marginal-dose challenges with SIVmac239, vaccinees were not protected from SIV acquisition but manifested partial control of viremia. Strikingly, the animal with the low-titer vaccine-induced anti-SIVmac239 NAb response acquired infection after the first SIVmac239 exposure. Collectively, these results highlight the difficulties in eliciting protective immunity against immunodeficiency virus infection.IMPORTANCE Our results are relevant to HIV vaccine development efforts because they suggest that increasing the number of booster immunizations or delivering additional viral antigens may not necessarily improve vaccine efficacy against immunodeficiency virus infection.

Vaccine protection against rectal acquisition of SIVmac239 in rhesus macaques.

A prophylactic vaccine against human immunodeficiency virus (HIV) remains a top priority in biomedical research. Given the failure of conventional immunization protocols to confer robust protection against HIV, new and unconventional approaches may be needed to generate protective anti-HIV immunity. Here we vaccinated rhesus macaques (RMs) with a recombinant (r)DNA prime (without any exogenous adjuvant), followed by a booster with rhesus monkey rhadinovirus (RRV)-a herpesvirus that establishes persistent infection in RMs (Group 1). Both the rDNA and rRRV vectors encoded a near-full-length simian immunodeficiency virus (SIVnfl) genome that assembles noninfectious SIV particles and expresses all nine SIV gene products. This rDNA/rRRV-SIVnfl vaccine regimen induced persistent anti-Env antibodies and CD8+ T-cell responses against the entire SIV proteome. Vaccine efficacy was assessed by repeated, marginal-dose, intrarectal challenges with SIVmac239. Encouragingly, vaccinees in Group 1 acquired SIVmac239 infection at a significantly delayed rate compared to unvaccinated controls (Group 3). In an attempt to improve upon this outcome, a separate group of rDNA/rRRV-SIVnfl-vaccinated RMs (Group 2) was treated with a cytotoxic T-lymphocyte antigen-4 (CTLA-4)-blocking monoclonal antibody during the vaccine phase and then challenged in parallel with Groups 1 and 3. Surprisingly, Group 2 was not significantly protected against SIVmac239 infection. In sum, SIVnfl vaccination can protect RMs against rigorous mucosal challenges with SIVmac239, a feat that until now had only been accomplished by live-attenuated strains of SIV. Further work is needed to identify the minimal requirements for this protection and whether SIVnfl vaccine efficacy can be improved by means other than anti-CTLA-4 adjuvant therapy.

The Frequency of Vaccine-Induced T-Cell Responses Does Not Predict the Rate of Acquisition after Repeated Intrarectal SIVmac239 Challenges in Mamu-B*08+ Rhesus Macaques.

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8+ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08-positive (Mamu-B*08+ ) RMs. Here we evaluated if robust vaccine-elicited CD8+ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08+ RMs following mucosal SIV challenges. Ten Mamu-B*08+ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8+ T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8+ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8+ T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

A recombinant herpesviral vector containing a near-full-length SIVmac239 genome produces SIV particles and elicits immune responses to all nine SIV gene products.

The properties of the human immunodeficiency virus (HIV) pose serious difficulties for the development of an effective prophylactic vaccine. Here we describe the construction and characterization of recombinant (r), replication-competent forms of rhesus monkey rhadinovirus (RRV), a gamma-2 herpesvirus, containing a near-full-length (nfl) genome of the simian immunodeficiency virus (SIV). A 306-nucleotide deletion in the pol gene rendered this nfl genome replication-incompetent as a consequence of deletion of the active site of the essential reverse transcriptase enzyme. Three variations were constructed to drive expression of the SIV proteins: one with SIV's own promoter region, one with a cytomegalovirus (cmv) immediate-early promoter/enhancer region, and one with an RRV dual promoter (p26 plus PAN). Following infection of rhesus fibroblasts in culture with these rRRV vectors, synthesis of the early protein Nef and the late structural proteins Gag and Env could be demonstrated. Expression levels of the SIV proteins were highest with the rRRV-SIVcmv-nfl construct. Electron microscopic examination of rhesus fibroblasts infected with rRRV-SIVcmv-nfl revealed numerous budding and mature SIV particles and these infected cells released impressive levels of p27 Gag protein (>150 ng/ml) into the cell-free supernatant. The released SIV particles were shown to be incompetent for replication. Monkeys inoculated with rRRV-SIVcmv-nfl became persistently infected, made readily-detectable antibodies against SIV, and developed T-cell responses against all nine SIV gene products. Thus, rRRV expressing a near-full-length SIV genome mimics live-attenuated strains of SIV in several important respects: the infection is persistent; >95% of the SIV proteome is naturally expressed; SIV particles are formed; and CD8+ T-cell responses are maintained indefinitely in an effector-differentiated state. Although the magnitude of anti-SIV immune responses in monkeys infected with rRRV-SIVcmv-nfl falls short of what is seen with live-attenuated SIV infection, further experimentation seems warranted.

Mamu-B*17+ Rhesus Macaques Vaccinated with env, vif, and nef Manifest Early Control of SIVmac239 Replication.

Certain major histocompatibility complex class I (MHC-I) alleles are associated with spontaneous control of viral replication in human immunodeficiency virus (HIV)-infected people and simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). These cases of "elite" control of HIV/SIV replication are often immune-mediated, thereby providing a framework for studying anti-lentiviral immunity. In this study, we examined how vaccination impacts SIV replication in RMs expressing the MHC-I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ and 50% of Mamu-B*08+ RMs control chronic-phase viremia after SIVmac239 infection. Because CD8+ T cells targeting Mamu-B*08-restricted SIV epitopes have been implicated in virologic suppression in Mamu-B*08+ RMs, we investigated whether this might also be true for Mamu-B*17+ RMs. Two groups of Mamu-B*17+ RMs were vaccinated with genes encoding Mamu-B*17-restricted epitopes in Vif and Nef. These genes were delivered by themselves (group 1) or together with env (group 2). Group 3 included MHC-I-matched RMs and served as the control group. Surprisingly, the group 1 vaccine regimen had little effect on viral replication compared to group 3, suggesting that unlike Mamu-B*08+ RMs, preexisting SIV-specific CD8+ T cells alone do not facilitate long-term virologic suppression in Mamu-B*17+ RMs. Remarkably, however, 5/8 group 2 vaccinees controlled viremia to <15 viral RNA copies/ml soon after infection. No serological neutralizing activity against SIVmac239 was detected in group 2, although vaccine-elicited gp140-binding antibodies correlated inversely with nadir viral loads. Collectively, these data shed new light on the unique mechanism of elite control in Mamu-B*17+ RMs and implicate vaccine-induced, nonneutralizing anti-Env antibodies in the containment of immunodeficiency virus infection.IMPORTANCE A better understanding of the immune correlates of protection against HIV might facilitate the development of a prophylactic vaccine. Therefore, we investigated simian immunodeficiency virus (SIV) infection outcomes in rhesus macaques expressing the major histocompatibility complex class I allele Mamu-B*17 Approximately 21% of Mamu-B*17+ macaques spontaneously controlled chronic phase viremia after SIV infection, an effect that may involve CD8+ T cells targeting Mamu-B*17-restricted SIV epitopes. We vaccinated Mamu-B*17+ macaques with genes encoding immunodominant epitopes in Vif and Nef alone (group 1) or together with env (group 2). Although neither vaccine regimen prevented SIV infection, 5/8 group 2 vaccinees controlled viremia to below detection limits shortly after infection. This outcome, which was not observed in group 1, was associated with vaccine-induced, nonneutralizing Env-binding antibodies. Together, these findings suggest a limited contribution of Vif- and Nef-specific CD8+ T cells for virologic control in Mamu-B*17+ macaques and implicate anti-Env antibodies in containment of SIV infection.

Vaccine-induced immune responses against both Gag and Env improve control of simian immunodeficiency virus replication in rectally challenged rhesus macaques.

The ability to control lentivirus replication may be determined, in part, by the extent to which individual viral proteins are targeted by the immune system. Consequently, defining the antigens that elicit the most protective immune responses may facilitate the design of effective HIV-1 vaccines. Here we vaccinated four groups of rhesus macaques with a heterologous vector prime/boost/boost/boost (PBBB) regimen expressing the following simian immunodeficiency virus (SIV) genes: env, gag, vif, rev, tat, and nef (Group 1); env, vif, rev, tat, and nef (Group 2); gag, vif, rev, tat, and nef (Group 3); or vif, rev, tat, and nef (Group 4). Following repeated intrarectal challenges with a marginal dose of the neutralization-resistant SIVmac239 clone, vaccinees in Groups 1-3 became infected at similar rates compared to control animals. Unexpectedly, vaccinees in Group 4 became infected at a slower pace than the other animals, although this difference was not statistically significant. Group 1 exhibited the best post-acquisition virologic control of SIV infection, with significant reductions in both peak and chronic phase viremia. Indeed, 5/8 Group 1 vaccinees had viral loads of less than 2,000 vRNA copies/mL of plasma in the chronic phase. Vaccine regimens that did not contain gag (Group 2), env (Group 3), or both of these inserts (Group 4) were largely ineffective at decreasing viremia. Thus, vaccine-induced immune responses against both Gag and Env appeared to maximize control of immunodeficiency virus replication. Collectively, these findings are relevant for HIV-1 vaccine design as they provide additional insights into which of the lentiviral proteins might serve as the best vaccine immunogens.

Potent Plasmablast-Derived Antibodies Elicited by the National Institutes of Health Dengue Vaccine.

Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes develops only after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long-sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. In this study, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan Institute. Eleven days after vaccination, we observed an ∼70-fold expansion of the plasmablast population. We generated 21 monoclonal Abs (MAbs) from singly sorted plasmablasts. These MAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen MAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 µg/ml or less; 6 of the 21 MAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing MAbs (nMAbs). Furthermore, the P3D05 nMAb neutralized DENV3 with extraordinary potency (concentration to achieve half-maximal neutralization [Neut50] = 0.03 µg/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that preexisting responses elicited by a previous DENV3 infection were recalled by immunization.IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut50 < 0.1 µg/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in phase III trials.

Vaccine-induced CD8+ T cells control AIDS virus replication.

Developing a vaccine for human immunodeficiency virus (HIV) may be aided by a complete understanding of those rare cases in which some HIV-infected individuals control replication of the virus. Most of these elite controllers express the histocompatibility alleles HLA-B*57 or HLA-B*27 (ref. 3). These alleles remain by far the most robust associations with low concentrations of plasma virus, yet the mechanism of control in these individuals is not entirely clear. Here we vaccinate Indian rhesus macaques that express Mamu-B*08, an animal model for HLA-B*27-mediated elite control, with three Mamu-B*08-restricted CD8(+) T-cell epitopes, and demonstrate that these vaccinated animals control replication of the highly pathogenic clonal simian immunodeficiency virus (SIV) mac239 virus. High frequencies of CD8(+) T cells against these Vif and Nef epitopes in the blood, lymph nodes and colon were associated with viral control. Moreover, the frequency of the CD8(+) T-cell response against the Nef RL10 epitope (Nef amino acids 137-146) correlated significantly with reduced acute phase viraemia. Finally, two of the eight vaccinees lost control of viral replication in the chronic phase, concomitant with escape in all three targeted epitopes, further implicating these three CD8(+) T-cell responses in the control of viral replication. Our findings indicate that narrowly targeted vaccine-induced virus-specific CD8(+) T-cell responses can control replication of the AIDS virus.

Dengue Virus Evades AAV-Mediated Neutralizing Antibody Prophylaxis in Rhesus Monkeys.

Development of vaccines against mosquito-borne Flaviviruses is complicated by the occurrence of antibody-dependent enhancement (ADE), which can increase disease severity. Long-term delivery of neutralizing antibodies (nAbs) has the potential to effectively block infection and represents an alternative to vaccination. The risk of ADE may be avoided by using prophylactic nAbs harboring amino acid mutations L234A and L235A (LALA) in the immunoglobulin G (IgG) constant region. Here, we used recombinant adeno-associated viruses (rAAVs) to deliver the anti-dengue virus 3 (DENV3) nAb P3D05. While the administration of rAAV-P3D05-rhesus immunoglobulin G1 (rhIgG1)-LALA to rhesus macaques engendered DENV3-neutralizing activity in serum, it did not prevent infection. The emergence of viremia following DENV3 challenge was delayed by 3-6 days in the rAAV-treated group, and replicating virus contained the envelope mutation K64R. This neutralization-resistant variant was also confirmed by virus outgrowth experiments in vitro. By delivering P3D05 with unmutated Fc sequences, we further demonstrated that DENV3 also evaded wild-type nAb prophylaxis, and serum viral loads appeared to be higher in the presence of low levels of unmutated P3D05-rhIgG1. Our study shows that a vectored approach for long-term delivery of nAbs with the LALA mutations is promising, but prophylaxis using a single nAb is likely insufficient at preventing DENV infection and replication.

Nef Is Dispensable for Resistance of Simian Immunodeficiency Virus-Infected Macrophages to CD8+ T Cell Killing.

UNLABELLED: Simian immunodeficiency virus (SIV)-specific CD8(+) T cells kill SIV-infected CD4(+) T cells in an major histocompatibility complex class I (MHC-I)-dependent manner. However, they are reportedly less efficient at killing SIV-infected macrophages. Since the viral accessory protein Nef has been shown to downregulate MHC-I molecules and enhance cytotoxic T lymphocyte (CTL) evasion in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells, we examined whether Nef played a role in protecting SIV-infected macrophages from killing by SIV-specific CD8(+) T cells. To explore the role of Nef in CD8(+) T cell evasion, we compared the ability of freshly sorted SIV-specific CD8(+) T cells to readily suppress viral replication or eliminate CD4(+) T cells or monocyte-derived macrophages infected with SIV variants containing wild-type (WT) or mutated nef genes. As expected, SIV-specific CD8(+) T cells suppressed viral replication and eliminated the majority of SIV-infected CD4(+) T cells, and this killing was enhanced in CD4(+) T cells infected with the nef variants. However, macrophages infected with nef variants that disrupt MHC-I downregulation did not promote rapid killing by freshly isolated CD8(+) T cells. These results suggest that mechanisms other than Nef-mediated MHC-I downregulation govern the resistance of SIV-infected macrophages to CD8(+) T cell-mediated killing. This study has implications for viral persistence and suggests that macrophages may afford primate lentiviruses some degree of protection from immune surveillance. IMPORTANCE: Myeloid cells are permissive for HIV/SIV replication in vitro and may contribute to viral persistence in vivo. While many studies have been geared to understanding how CD8(+) T cells control viral replication in CD4(+) T cells, the role of these cells in controlling viral replication in macrophages is less clear. Primary, unstimulated CD8(+) T cells insignificantly suppress viral replication or eliminate SIV-infected macrophages. Since the viral Nef protein downregulates MHC-I and provides infected cells some degree of protection from CD8(+) T cell-mediated effector functions, we evaluated whether Nef may be contributing to the resistance of macrophages to CD8(+) T cell suppression. Our results suggest that Nef is not involved in protecting infected macrophages from CD8(+) T cell killing and suggest that other mechanisms are involved in macrophage evasion from CD8 surveillance.

Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8+ T-Cell Responses Focused on a Single Nef Epitope Select for Escape Variants Shortly after Infection.

UNLABELLED: Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08(+) animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08(+) macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8(+) T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8(+) T-cell response would facilitate the development of elite control in Mamu-B*08(+) animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08(+) animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8(+) T cells. These vaccine-induced effector memory CD8(+) T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8(+) T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08(+) macaques. IMPORTANCE: Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8(+) T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08(+) rhesus macaques­a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8(+) T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08(+) monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8(+) T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8(+) T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection.

Vaccination with Gag, Vif, and Nef gene fragments affords partial control of viral replication after mucosal challenge with SIVmac239.

UNLABELLED: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01(+) vaccinees mounted CD8(+) T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE: Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8(+) T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01(+) vaccinees. This finding underscores the difficulty of inducing subdominant CD8(+) T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.

The majority of freshly sorted simian immunodeficiency virus (SIV)-specific CD8(+) T cells cannot suppress viral replication in SIV-infected macrophages.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) primarily infect activated CD4(+) T cells but can infect macrophages. Surprisingly, ex vivo tetramer-sorted SIV-specific CD8(+) T cells that eliminated and suppressed viral replication in SIV-infected CD4(+) T cells failed to do so in SIV-infected macrophages. It is possible, therefore, that while AIDS virus-infected macrophages constitute only a small percentage of all virus-infected cells, they may be relatively resistant to CD8(+) T cell-mediated lysis and continue to produce virus over long periods of time.

Adeno-associated virus-vectored delivery of HIV biologics: the promise of a "single-shot" functional cure for HIV infection.

The ability of immunoglobulin-based HIV biologics (Ig-HIV), including broadly neutralizing antibodies, to suppress viral replication in pre-clinical and clinical studies illustrates how these molecules can serve as alternatives or adjuncts to antiretroviral therapy for treating HIV infection. However, the current paradigm for delivering Ig-HIVs requires repeated passive infusions, which faces both logistical and economic challenges to broad-scale implementation. One promising way to overcome these obstacles and achieve sustained expression of Ig-HIVs in vivo involves the transfer of Ig-HIV genes to host cells utilizing adeno-associated virus (AAV) vectors. Because AAV vectors are non-pathogenic and their genomes persist in the cell nucleus as episomes, transgene expression can last for as long as the AAV-transduced cell lives. Given the long lifespan of myocytes, skeletal muscle is a preferred tissue for AAV-based immunotherapies aimed at achieving persistent delivery of Ig-HIVs. Consistent with this idea, recent studies suggest that lifelong immunity against HIV can be achieved from a one-time intramuscular dose of AAV/Ig-HIV vectors. However, realizing the promise of this approach faces significant hurdles, including the potential of AAV-delivered Ig-HIVs to induce anti-drug antibodies and the high AAV seroprevalence in the human population. Here we describe how these host immune responses can hinder AAV/Ig-HIV therapies and review current strategies for overcoming these barriers. Given the potential of AAV/Ig-HIV therapy to maintain ART-free virologic suppression and prevent HIV reinfection in people living with HIV, optimizing this strategy should become a greater priority in HIV/AIDS research.

Dengue virus-specific CD4+ and CD8+ T lymphocytes target NS1, NS3 and NS5 in infected Indian rhesus macaques.

Every year, Dengue virus (DENV) infects approximately 100 million people. There are currently several vaccines undergoing clinical studies, but most target the induction of neutralizing antibodies. Unfortunately, DENV infection can be enhanced by subneutralizing levels of antibodies that bind virions and deliver them to cells of the myeloid lineage, thereby increasing viral replication (termed antibody-dependent enhancement [ADE]). T lymphocyte-based vaccines may offer an alternative that avoids ADE. The goal of our study was to describe the cellular immune response generated after primary DENV infection in Indian rhesus macaques. We infected eight rhesus macaques with 105 plaque-forming units (PFU) of DENV serotype 2 (DENV2) New Guinea C (NGC) strain, and monitored viral load and the cellular immune response to the virus. Viral replication peaked at day 4 post-infection and was resolved by day 10. DENV-specific CD4+ and CD8+ T lymphocytes targeted nonstructural (NS) 1, NS3 and NS5 proteins after resolution of peak viremia. DENV-specific CD4+ cells expressed interferon-gamma (IFN-γ) along with tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and macrophage inflammatory protein-1 beta (MIP-1ß). In comparison, DENV-specific CD8+ cells expressed IFN-γ in addition to MIP-1ß and TNF-α and were positive for the degranulation marker CD107a. Interestingly, a fraction of the DENV-specific CD4+ cells also stained for CD107a, suggesting that they might be cytotoxic. Our results provide a more complete understanding of the cellular immune response during DENV infection in rhesus macaques and contribute to the development of rhesus macaques as an animal model for DENV vaccine and pathogenicity studies.

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection.

The precise immunological role played by CD4(+) T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8(+) cell depletion, elite controlling macaques with set-point viral loads < or = 500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4(+) T cell responses during reestablishment of control with > or = 54% of all virus-specific CD4(+) T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4(+) T cells neither recognized nor suppressed viral replication in SIV-infected CD4(+) T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4(+) T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4(+) T cells may contribute directly to elite control by inhibiting viral replication in macrophages.

T-cell correlates of vaccine efficacy after a heterologous simian immunodeficiency virus challenge.

Determining the "correlates of protection" is one of the challenges in human immunodeficiency virus vaccine design. To date, T-cell-based AIDS vaccines have been evaluated with validated techniques that measure the number of CD8(+) T cells in the blood that secrete cytokines, mainly gamma interferon (IFN-gamma), in response to synthetic peptides. Despite providing accurate and reproducible measurements of immunogenicity, these methods do not directly assess antiviral function and thus may not identify protective CD8(+) T-cell responses. To better understand the correlates of vaccine efficacy, we analyzed the immune responses elicited by a successful T-cell-based vaccine against a heterologous simian immunodeficiency virus challenge. We searched for correlates of protection using a viral suppression assay (VSA) and an IFN-gamma enzyme-linked immunospot assay. While the VSA measured in vitro suppression, it did not predict the outcome of the vaccine trial. However, we found several aspects of the vaccine-induced T-cell response that were associated with improved outcome after challenge. Of note, broad vaccine-induced prechallenge T-cell responses directed against Gag and Vif correlated with lower viral loads and higher CD4(+) lymphocyte counts. These results may be relevant for the development of T-cell-based AIDS vaccines since they indicate that broad epitope-specific repertoires elicited by vaccination might serve as a correlate of vaccine efficacy. Furthermore, the present study demonstrates that certain viral proteins may be more effective than others as vaccine immunogens.