Variation within major internal repeats of KSHV in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), yet the viral genetic factors that lead to the development of KS in KSHV-infected individuals have not been fully elucidated. Nearly, all previous analyses of KSHV genomic evolution and diversity have excluded the three major internal repeat regions: the two origins of lytic replication, internal repeats 1 and 2 (IR1 and IR2), and the latency-associated nuclear antigen (LANA) repeat domain (LANAr). These regions encode protein domains that are essential to the KSHV infection cycle but have been rarely sequenced due to their extended repetitive nature and high guanine and cytosine (GC) content. The limited data available suggest that their sequences and repeat lengths are more heterogeneous across individuals than in the remainder of the KSHV genome. To assess their diversity, the full-length IR1, IR2, and LANAr sequences, tagged with unique molecular identifiers (UMIs), were obtained by Pacific Biosciences' single-molecule real-time sequencing (SMRT-UMI) from twenty-four tumors and six matching oral swabs from sixteen adults in Uganda with advanced KS. Intra-host single-nucleotide variation involved an average of 0.16 per cent of base positions in the repeat regions compared to a nearly identical average of 0.17 per cent of base positions in the remainder of the genome. Tandem repeat unit (TRU) counts varied by only one from the intra-host consensus in a majority of individuals. Including the TRU indels, the average intra-host pairwise identity was 98.3 per cent for IR1, 99.6 per cent for IR2 and 98.9 per cent for LANAr. More individuals had mismatches and variable TRU counts in IR1 (twelve/sixteen) than in IR2 (two/sixteen). There were no open reading frames in the Kaposin coding sequence inside IR2 in at least fifty-five of ninety-six sequences. In summary, the KSHV major internal repeats, like the rest of the genome in individuals with KS, have low diversity. IR1 was the most variable among the repeats, and no intact Kaposin reading frames were present in IR2 of the majority of genomes sampled.

Pharmacokinetic serum concentrations of VRC01 correlate with prevention of HIV-1 acquisition.

BACKGROUND: The phase 2b proof-of-concept Antibody Mediated Prevention (AMP) trials showed that VRC01, an anti-HIV-1 broadly neutralising antibody (bnAb), prevented acquisition of HIV-1 sensitive to VRC01. To inform future study design and dosing regimen selection of candidate bnAbs, we investigated the association of VRC01 serum concentration with HIV-1 acquisition using AMP trial data. METHODS: The case-control sample included 107 VRC01 recipients who acquired HIV-1 and 82 VRC01 recipients who remained without HIV-1 during the study. We measured VRC01 serum concentrations with a qualified pharmacokinetic (PK) Binding Antibody Multiplex Assay. We employed nonlinear mixed effects PK modelling to estimate daily-grid VRC01 concentrations. Cox regression models were used to assess the association of VRC01 concentration at exposure and baseline body weight, with the hazard of HIV-1 acquisition and prevention efficacy as a function of VRC01 concentration. We also compared fixed dosing vs. body weight-based dosing via simulations. FINDINGS: Estimated VRC01 concentrations in VRC01 recipients without HIV-1 were higher than those in VRC01 recipients who acquired HIV-1. Body weight was inversely associated with HIV-1 acquisition among both placebo and VRC01 recipients but did not modify the prevention efficacy of VRC01. VRC01 concentration was inversely correlated with HIV-1 acquisition, and positively correlated with prevention efficacy of VRC01. Simulation studies suggest that fixed dosing may be comparable to weight-based dosing in overall predicted prevention efficacy. INTERPRETATION: These findings suggest that bnAb serum concentration may be a useful marker for dosing regimen selection, and operationally efficient fixed dosing regimens could be considered for future trials of HIV-1 bnAbs. FUNDING: Was provided by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) (UM1 AI068614, to the HIV Vaccine Trials Network [HVTN]; UM1 AI068635, to the HVTN Statistical Data and Management Center [SDMC], Fred Hutchinson Cancer Center [FHCC]; 2R37 054165 to the FHCC; UM1 AI068618, to HVTN Laboratory Center, FHCC; UM1 AI068619, to the HPTN Leadership and Operations Center; UM1 AI068613, to the HIV Prevention Trials Network [HPTN] Laboratory Center; UM1 AI068617, to the HPTN SDMC; and P30 AI027757, to the Center for AIDS Research, Duke University (AI P30 AI064518) and University of Washington (P30 AI027757) Centers for AIDS Research; R37AI054165 from NIAID to the FHCC; and OPP1032144 CA-VIMC Bill & Melinda Gates Foundation.

Genomic changes in Kaposi Sarcoma-associated Herpesvirus and their clinical correlates.

Kaposi sarcoma (KS), a common HIV-associated malignancy, presents a range of clinicopathological features. Kaposi sarcoma-associated herpesvirus (KSHV) is its etiologic agent, but the contribution of viral genomic variation to KS development is poorly understood. To identify potentially influential viral polymorphisms, we characterized KSHV genetic variation in 67 tumors from 1-4 distinct sites from 29 adults with advanced KS in Kampala, Uganda. Whole KSHV genomes were sequenced from 20 tumors with the highest viral load, whereas only polymorphic genes were screened by PCR and sequenced from 47 other tumors. Nine individuals harbored ≥1 tumors with a median 6-fold over-coverage of a region centering on K5 and K6 genes. K8.1 gene was inactivated in 8 individuals, while 5 had mutations in the miR-K10 microRNA coding sequence. Recurring inter-host polymorphisms were detected in K4.2 and K11.2. The K5-K6 region rearrangement breakpoints and K8.1 mutations were all unique, indicating that they arise frequently de novo. Rearrangement breakpoints were associated with potential G-quadruplex and Z-DNA forming sequences. Exploratory evaluations of viral mutations with clinical and tumor traits were conducted by logistic regression without multiple test corrections. K5-K6 over-coverage and K8.1 inactivation were tentatively correlated (p<0.001 and p = 0.005, respectively) with nodular rather than macular tumors, and with individuals that had lesions in ≤4 anatomic areas (both p≤0.01). Additionally, a trend was noted for miR-K10 point mutations and lower survival rates (HR = 4.11, p = 0.053). Two instances were found of distinct tumors within an individual sharing the same viral mutation, suggesting metastases or transmission of the aberrant viruses within the host. To summarize, KSHV genomes in tumors frequently have over-representation of the K5-K6 region, as well as K8.1 and miR-K10 mutations, and each might be associated with clinical phenotypes. Studying their possible effects may be useful for understanding KS tumorigenesis and disease progression.

In-depth single-cell analysis of translation-competent HIV-1 reservoirs identifies cellular sources of plasma viremia.

Clonal expansion of HIV-infected cells contributes to the long-term persistence of the HIV reservoir in ART-suppressed individuals. However, the contribution from cell clones that harbor inducible proviruses to plasma viremia is poorly understood. Here, we describe a single-cell approach to simultaneously sequence the TCR, integration sites and proviral genomes from translation-competent reservoir cells, called STIP-Seq. By applying this approach to blood samples from eight participants, we show that the translation-competent reservoir mainly consists of proviruses with short deletions at the 5'-end of the genome, often involving the major splice donor site. TCR and integration site sequencing reveal that cell clones with predicted pathogen-specificity can harbor inducible proviruses integrated into cancer-related genes. Furthermore, we find several matches between proviruses retrieved with STIP-Seq and plasma viruses obtained during ART and upon treatment interruption, suggesting that STIP-Seq can capture clones that are responsible for low-level viremia or viral rebound.

Intra-host changes in Kaposi sarcoma-associated herpesvirus genomes in Ugandan adults with Kaposi sarcoma.

Intra-host tumor virus variants may influence the pathogenesis and treatment responses of some virally-associated cancers. However, the intra-host variability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma (KS), has to date been explored with sequencing technologies that possibly introduce more errors than that which occurs in the viral population, and these studies have only studied variable regions. Here, full-length KSHV genomes in tumors and/or oral swabs from 9 Ugandan adults with HIV-associated KS were characterized. Furthermore, we used deep, short-read sequencing using duplex unique molecular identifiers (dUMI)-random double-stranded oligonucleotides that barcode individual DNA molecules before library amplification. This allowed suppression of PCR and sequencing errors to ~10-9/base as well as afforded accurate determination of KSHV genome numbers sequenced in each sample. KSHV genomes were assembled de novo, and rearrangements observed were confirmed by PCR and Sanger sequencing. 131-kb KSHV genome sequences, excluding major repeat regions, were successfully obtained from 23 clinical specimens, averaging 2.3x104 reads/base. Strikingly, KSHV genomes were virtually identical within individuals at the point mutational level. The intra-host heterogeneity that was observed was confined to tumor-associated KSHV mutations and genome rearrangements, all impacting protein-coding sequences. Although it is unclear whether these changes were important to tumorigenesis or occurred as a result of genomic instability in tumors, similar changes were observed across individuals. These included inactivation of the K8.1 gene in tumors of 3 individuals and retention of a region around the first major internal repeat (IR1) in all instances of genomic deletions and rearrangements. Notably, the same breakpoint junctions were found in distinct tumors within single individuals, suggesting metastatic spread of rearranged KSHV genomes. These findings define KSHV intra-host heterogeneity in vivo with greater precision than has been possible in the past and suggest the possibility that aberrant KSHV genomes may contribute to aspects of KS tumorigenesis. Furthermore, study of KSHV with use of dUMI provides a proof of concept for utilizing this technique for detailed study of other virus populations in vivo.

Phylogenetic Analyses Comparing HIV Sequences from Plasma at Virologic Failure to Cervix Versus Blood Sequences from Antecedent Antiretroviral Therapy Suppression.

Identifying tissue sources of HIV that rebound following "failure" of antiretroviral therapy (ART) is critical to evaluating cure strategies. To assess the role of the uterine cervix and peripheral blood mononuclear cells (PBMC) as viral reservoirs, nearest-neighbor phylogenetic analyses compared genetic relatedness of tissue sequences during ART suppression to those detected in plasma at viral rebound. Blood and genital tract specimens from a natural history cohort of HIV-infected women were collected over 5 years. HIV DNA sequences extracted from PBMC and cervical biopsies during ART suppression and plasma RNA from rebound (defined as HIV RNA >3 log10 copies/mL) were derived by single-genome amplification. Phylogenetic and nearest-neighbor analyses of HIV env sequences and drug resistance in pol sequences were compared between tissues. Nine instances of plasma viral rebound (median HIV RNA 3.6 log10 c/mL; IQR: 3.1-3.8) were detected in 7 of 57 women. Nearest-neighbor analyses found rebound plasma sequences were closer to uterine cervical sequences in 4/9 (44%), closer to PBMC in 3/9 (33%), and ambiguous in 2/9 (22%) cases. Rebound plasma clades (n = 27) shared identical sequences in seven instances with the cervix versus two with PBMC. Novel drug resistance mutations were detected in 4/9 (44%) rebounds. The observed tendency for greater sharing of identical HIV variants and greater nearest-neighbor association between rebounding plasma and uterine cervical versus PBMC sequences suggests that the uterine cervix may be a relevant HIV reservoir. The cervix, a readily accessible tissue in women that can be repeatedly sampled, could help assess the HIV reservoir when evaluating cure strategies.

Gag and env conserved element CE DNA vaccines elicit broad cytotoxic T cell responses targeting subdominant epitopes of HIV and SIV Able to recognize virus-infected cells in macaques.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting inessential variable regions are hurdles in the development of an effective AIDS vaccine. We developed a DNA vaccine comprising conserved elements (CE) of SIV p27Gag and HIV-1 Env and found that priming vaccination with CE DNA is critical to efficiently overcome the dominance imposed by Gag and Env variable regions. Here, we show that DNA vaccinated macaques receiving the CE prime/CE+full-length DNA co-delivery booster vaccine regimens developed broad, potent and durable cytotoxic T cell responses targeting conserved protein segments of SIV Gag and HIV Env. Gag CE-specific T cells showed robust anamnestic responses upon infection with SIVmac239 which led to the identification of CE-specific cytotoxic lymphocytes able to recognize epitopes covering distinct CE on the surface of SIV infected cells in vivo. Though not controlling infection overall, we found an inverse correlation between Gag CE-specific CD8+ T cell responses and peak viremia. The T cell responses induced by the HIV Env CE immunogen were recalled in some animals upon SIV infection, leading to the identification of two cross-reactive epitopes between HIV and SIV Env based in sequence homology. These data demonstrate that a vaccine combining Gag and Env CE DNA subverted the normal immunodominance patterns, eliciting immune responses that included subdominant, highly conserved epitopes. These vaccine regimens augment cytotoxic T cell responses to highly conserved epitopes in the viral proteome and maximize response breadth. The vaccine-induced CE-specific T cells were expanded upon SIV infection, indicating that the predicted CE epitopes incorporated in the DNA vaccine are processed and exposed by infected cells in their natural context within the viral proteome.

DNA Vaccine-Induced Long-Lasting Cytotoxic T Cells Targeting Conserved Elements of Human Immunodeficiency Virus Gag Are Boosted Upon DNA or Recombinant Modified Vaccinia Ankara Vaccination.

DNA-based vaccines able to induce efficient cytotoxic T-cell responses targeting conserved elements (CE) of human immunodeficiency virus type 1 (HIV-1) Gag have been developed. These CE were selected by stringent conservation, the ability to induce T-cell responses with broad human leukocyte antigen coverage, and the association between recognition of CE epitopes and viral control in HIV-infected individuals. Based on homology to HIV, a simian immunodeficiency virus p27gag CE DNA vaccine has also been developed. This study reports on the durability of the CE-specific T-cell responses induced by HIV and simian immunodeficiency virus CE DNA-based prime/boost vaccine regimens in rhesus macaques, and shows that the initially primed CE-specific T-cell responses were efficiently boosted by a single CE DNA vaccination after the long rest period (up to 2 years). In another cohort of animals, the study shows that a single inoculation with non-replicating recombinant Modified Vaccinia Ankara (rMVA62B) also potently boosted CE-specific responses after around 1.5 years of rest. Both CE DNA and rMVA62B booster vaccinations increased the magnitude and cytotoxicity of the CE-specific responses while maintaining the breadth of CE recognition. Env produced by rMVA62B did not negatively interfere with the recall of the Gag CE responses. rMVA62B could be beneficial to further boosting the immune response to Gag in humans. Vaccine regimens that employ CE DNA as a priming immunogen hold promise for application in HIV prevention and therapy.

Therapeutic conserved elements (CE) DNA vaccine induces strong T-cell responses against highly conserved viral sequences during simian-human immunodeficiency virus infection.

HIV-specific T-cell responses play a key role in controlling HIV infection, and therapeutic vaccines for HIV that aim to improve viral control will likely need to improve on the T-cell responses induced by infection. However, in the setting of chronic infection, an effective therapeutic vaccine must overcome the enormous viral genetic diversity and the presence of pre-existing T-cell responses that are biased toward immunodominant T-cell epitopes that can readily mutate to evade host immunity and thus potentially provide inferior protection. To address these issues, we investigated a novel, epidermally administered DNA vaccine expressing SIV capsid (p27Gag) homologues of highly conserved elements (CE) of the HIV proteome in macaques experiencing chronic but controlled SHIV infection. We assessed the ability to boost or induce de novo T-cell responses against the conserved but immunologically subdominant CE epitopes. Two groups of animals were immunized with either the CE DNA vaccine or a full-length SIV p57gag DNA vaccine. Prior to vaccination, CE responses were similar in both groups. The full-length p57gag DNA vaccine, which contains the CE, increased overall Gag-specific responses but did not increase CE responses in any animals (0/4). In contrast, the CE DNA vaccine increased CE responses in all (4/4) vaccinated macaques. In SIV infected but unvaccinated macaques, those that developed stronger CE-specific responses during acute infection exhibited lower viral loads. We conclude that CE DNA vaccination can re-direct the immunodominance hierarchy towards CE in the setting of attenuated chronic infection and that induction of these responses by therapeutic vaccination may improve immune control of HIV.

Transmission of HIV-1 drug resistance mutations within partner-pairs: A cross-sectional study of a primary HIV infection cohort.

BACKGROUND: Transmission of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations, particularly that of minority drug-resistant variants, remains poorly understood. Population-based studies suggest that drug-resistant HIV-1 is less transmissible than drug-susceptible viruses. We compared HIV-1 drug-resistant genotypes among partner-pairs in order to assess the likelihood of transmission of drug resistance mutations and investigate the role of minority variants in HIV transmission. METHODS AND FINDINGS: From 1992-2010, 340 persons with primary HIV-1 infection and their partners were enrolled into observational research studies at the University of Washington Primary Infection Clinic (UWPIC). Out of 50 partner-pairs enrolled, 36 (72%) transmission relationships were confirmed by phylogenetic distance analysis of HIV-1 envelope (env) sequences, and 31 partner-pairs enrolled after 1995 met criteria for this study. Drug resistance mutations in the region of the HIV-1 polymerase gene (pol) that encodes protease and reverse transcriptase were assessed by 454-pyrosequencing. In 25 partner-pairs where the transmission direction could be determined, 12 (48%) transmitters had 1-4 drug resistance mutations (23 total) detected in their HIV-1 populations at a median frequency of 6.0% (IQR 1.5%-98.7%, range 1.0%-99.6%). Of 10 major mutations detected in five transmitters at a frequency >95%, 100% (95% CI 69.2%-100%) were detected in recipients. All of these transmitters were antiretroviral (ARV)-naïve at the time of specimen collection. Fourteen mutations (eight major mutations and six accessory mutations) were detected in nine transmitters at low frequencies (1.0%-11.8%); four of these transmitters had previously received ARV therapy. Two (14% [95% CI 1.8%-42.8%]) G73S accessory mutations were detected in both transmitter and recipient. This number is not significantly different from the number expected based on the observed frequencies of drug-resistant viruses in transmitting partners. Limitations of this study include the small sample size and uncertainties in determining the timing of virus transmission and mutation history. CONCLUSIONS: Drug-resistant majority variants appeared to be commonly transmitted by ARV-naïve participants in our analysis and may contribute significantly to transmitted drug resistance on a population level. When present at low frequency, no major mutation was observed to be shared between partner-pairs; identification of accessory mutations shared within a pair could be due to transmission, laboratory artifact, or apolipoprotein B mRNA-editing enzyme, catalytic polypeptides (APOBECs), and warrants further study.

HIV population-level adaptation can rapidly diminish the impact of a partially effective vaccine.

BACKGROUND: Development of an HIV vaccine might be essential to ending the HIV/AIDS pandemic. However, vaccines can result in the emergence and spread of vaccine-resistant strains. Indeed, analyses of breakthrough infections in the HIV phase 3 vaccine trial RV144 identified HIV genotypes with differential rates of transmission in vaccine and placebo recipients. We hypothesized that, for HIV vaccination programs based on partially effective vaccines similar to RV144, HIV adaptation will rapidly diminish the expected vaccine impact. METHODS AND FINDINGS: Using two HIV epidemic models, we simulated large-scale vaccination programs and, critically, included HIV strain diversity with respect to the vaccine response. We show here that rapid population-level viral adaptation can lead to decreased overall vaccine efficacy and substantially fewer infections averted by vaccination, when comparing scenarios with and without viral evolution (with outcomes depending on vaccination coverage, vaccine efficacy against the sensitive allele, and the initial resistant allele frequency). Translating this to the epidemic in South Africa, a scenario with 70% vaccination coverage may result in 250,000 infections (non-averted by vaccination) within 10 years of vaccine rollout that are due solely to HIV adaptation, all else being equal. CONCLUSIONS: These findings suggest that approaches to HIV vaccine development, program implementation, and epidemic modeling may require attention to viral adaptation in response to vaccination.

Sieve analysis of breakthrough HIV-1 sequences in HVTN 505 identifies vaccine pressure targeting the CD4 binding site of Env-gp120.

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector.

HIV Env conserved element DNA vaccine alters immunodominance in macaques.

Sequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of 'conserved' and 'variable' regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0-5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines.

Clonal Expansion of Human Immunodeficiency Virus-Infected Cells and Human Immunodeficiency Virus Persistence During Antiretroviral Therapy.

The latent HIV-1 reservoir in blood decays very slowly, even during prolonged suppression of viral replication by antiretroviral therapy (ART). Mechanisms for reservoir persistence include replenishment through low-level viral replication, longevity and homeostatic proliferation of memory T cells, and most recently appreciated, clonal expansion of HIV-infected cells. Clonally expanded cells make up a large and increasing fraction of the residual infected cell population on ART, and insertion of HIV proviruses into certain host cellular genes has been associated with this proliferation. That the vast majority of proviruses are defective clouds our assessment of the degree to which clonally expanded cells harbor infectious viruses, and thus the extent to which they contribute to reservoirs relevant to curing infection. This review summarizes past studies that have defined our current understanding and the gaps in our knowledge of the mechanisms by which proviral integration and clonal expansion sustain the HIV reservoir.

DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth.

Effective Cytotoxic T Lymphocyte Targeting of Persistent HIV-1 during Antiretroviral Therapy Requires Priming of Naive CD8+ T Cells.

UNLABELLED: Curing HIV-1 infection will require elimination of persistent cellular reservoirs that harbor latent virus in the face of combination antiretroviral therapy (cART). Proposed immunotherapeutic strategies to cure HIV-1 infection include enhancing lysis of these infected cells by cytotoxic T lymphocytes (CTL). A major challenge in this strategy is overcoming viral immune escape variants that have evaded host immune control. Here we report that naive CD8(+) T cells from chronic HIV-1-infected participants on long-term cART can be primed by dendritic cells (DC). These DC must be mature, produce high levels of interleukin 12p70 (IL-12p70), be responsive to CD40 ligand (CD40L), and be loaded with inactivated, autologous HIV-1. These DC-primed CD8(+) T cell responders produced high levels of gamma interferon (IFN-γ) in response to a broad range of both conserved and variable regions of Gag and effectively killed CD4(+) T cell targets that were either infected with the autologous latent reservoir-associated virus or loaded with autologous Gag peptides. In contrast, HIV-1-specific memory CD8(+) T cells stimulated with autologous HIV-1-loaded DC produced IFN-γ in response to a narrow range of conserved and variable Gag peptides compared to the primed T cells and most notably, displayed significantly lower cytolytic function. Our findings highlight the need to selectively induce new HIV-1-specific CTL from naive precursors while avoiding activation of existing, dysfunctional memory T cells in potential curative immunotherapeutic strategies for HIV-1 infection. IMPORTANCE: Current immunotherapeutic approaches aim to enhance antiviral immunity against the HIV-1 reservoir; however, it has yet to be shown whether T cells from persons on cART can recognize and eliminate virus-infected cells. We show that in persons on cART a personalized medicine approach using their dendritic cells to stimulate their naive T cells induces potent effector CTL in vitro that recognize and eradicate HIV-1-infected CD4(+) T cells. Additionally, we show that the same stimulation of existing memory T cells results in cytokine secretion but limited effector function. Our study demonstrates that the naive T cell repertoire can recognize persistent HIV-1 during cART and supports immunotherapy strategies for an HIV-1 cure that targets naive T cells, rather than existing, dysfunctional, memory T cells.

Composite Sequence-Structure Stability Models as Screening Tools for Identifying Vulnerable Targets for HIV Drug and Vaccine Development.

Rapid evolution and high sequence diversity enable Human Immunodeficiency Virus (HIV) populations to acquire mutations to escape antiretroviral drugs and host immune responses, and thus are major obstacles for the control of the pandemic. One strategy to overcome this problem is to focus drugs and vaccines on regions of the viral genome in which mutations are likely to cripple function through destabilization of viral proteins. Studies relying on sequence conservation alone have had only limited success in determining critically important regions. We tested the ability of two structure-based computational models to assign sites in the HIV-1 capsid protein (CA) that would be refractory to mutational change. The destabilizing mutations predicted by these models were rarely found in a database of 5811 HIV-1 CA coding sequences, with none being present at a frequency greater than 2%. Furthermore, 90% of variants with the low predicted stability (from a set of 184 CA variants whose replication fitness or infectivity has been studied in vitro) had aberrant capsid structures and reduced viral infectivity. Based on the predicted stability, we identified 45 CA sites prone to destabilizing mutations. More than half of these sites are targets of one or more known CA inhibitors. The CA regions enriched with these sites also overlap with peptides shown to induce cellular immune responses associated with lower viral loads in infected individuals. Lastly, a joint scoring metric that takes into account both sequence conservation and protein structure stability performed better at identifying deleterious mutations than sequence conservation or structure stability information alone. The computational sequence-structure stability approach proposed here might therefore be useful for identifying immutable sites in a protein for experimental validation as potential targets for drug and vaccine development.

Lack of viral control and development of combination antiretroviral therapy escape mutations in macaques after bone marrow transplantation.

OBJECTIVE: We have previously demonstrated robust control of simian/human immunodeficiency virus (SHIV1157-ipd3N4) viremia following administration of combination antiretroviral therapy (cART) in pigtailed macaques. Here, we sought to determine the safety of hematopoietic stem cell transplantation (HSCT) in cART-suppressed and unsuppressed animals. DESIGN: We compared disease progression in animals challenged with SHIV 100 days post-transplant, to controls that underwent transplant following SHIV challenge and stable cART-dependent viral suppression. METHODS: SHIV viral load, cART levels, and anti-SHIV antibodies were measured longitudinally from plasma/serum from each animal. Flow cytometry was used to assess T-cell subset frequencies in peripheral blood and the gastrointestinal tract. Deep sequencing was used to identify cART resistance mutations. RESULTS: In control animals, virus challenge induced transient peak viremia, viral set point, and durable suppression by cART. Subsequent HSCT was not associated with adverse events in these animals. Post-transplant animals were challenged during acute recovery following HSCT, and displayed sustained peak viremia and cART resistance. Although post-transplant animals had comparable plasma levels of antiretroviral drugs and showed no evidence of enhanced infection of myeloid subsets in the periphery, they exhibited a drastic reduction in virus-specific antibody production and decreased T-cell counts. CONCLUSIONS: These results suggest that virus challenge prior to complete transplant recovery impairs viral control and may promote drug resistance. These findings may also have implications for scheduled treatment interruption studies in patients on cART during post-HSCT recovery: premature scheduled treatment interruption could similarly result in lack of viral control and cART resistance.

Resistance detected by pyrosequencing following zidovudine monotherapy for prevention of HIV-1 mother-to-child-transmission.

To prevent mother-to-child-transmission of HIV-1, the 2010 WHO guidelines recommended prenatal zidovudine (ZDV) monotherapy (option A). To determine if ZDV monotherapy selects for HIV resistance in antiretroviral-naive women during pregnancy, specimens from 50 individuals were examined using pyrosequencing. ZDV-resistance mutations were detected at delivery in seven women (14%, 95% confidence interval 6.6-26.5%). These data raise the question whether women administered ZDV monotherapy for prevention of mother-to-child-transmission could have higher risk of virologic failure when later started on combination antiretroviral therapy, as has been demonstrated following single-dose nevirapine prophylaxis.

A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques.

BACKGROUND: None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs. METHODS: To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1. RESULTS: Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4(+) and CD8(+) T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4(+) and CD8(+) T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ(+) CD8(+) T cells being Granzyme B(+) and able to degranulate (CD107a(+)). CONCLUSIONS: These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.