Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

CD4 enumeration technologies: a systematic review of test performance for determining eligibility for antiretroviral therapy.

BACKGROUND: Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration. METHODS AND FINDINGS: Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained. CONCLUSIONS: A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.

Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.

Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation.

Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

A Brief Chronicle of CD4 as a Biomarker for HIV/AIDS: A Tribute to the Memory of John L. Fahey.

Foundational cellular immunology research of the 1960s and 1970s, together with the advent of monoclonal antibodies and flow cytometry, provided the knowledge base and the technological capability that enabled the elucidation of the role of CD4 T cells in HIV infection. Research identifying the sources and magnitude of variation in CD4 measurements, standardized reagents and protocols, and the development of clinical flow cytometers all contributed to the feasibility of widespread CD4 testing. Cohort studies and clinical trials provided the context for establishing the utility of CD4 for prognosis in HIV-infected persons, initial assessment of in vivo antiretroviral drug activity, and as a surrogate marker for clinical outcome in antiretroviral therapeutic trials. Even with sensitive HIV viral load measurement, CD4 cell counting is still utilized in determining antiretroviral therapy eligibility and time to initiate therapy. New point of care technologies are helping both to lower the cost of CD4 testing and enable its use in HIV test and treat programs around the world.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.

Potent and broad neutralizing activity of a single chain antibody fragment against cell-free and cell-associated HIV-1.

Several human monoclonal antibodies (hmAbs) exhibit relatively potent and broad neutralizing activity against HIV-1, but there has not been much success in using them as potential therapeutics. We have previously hypothesized and demonstrated that small engineered antibodies can target highly conserved epitopes that are not accessible by full-size antibodies. However, their potency has not been comparatively evaluated with known HIV-1-neutralizing hmAbs against large panels of primary isolates. We report here the inhibitory activity of an engineered single chain antibody fragment (scFv), m9, against several panels of primary HIV-1 isolates from group M (clades A-G) using cell-free and cell-associated virus in cell line-based assays. M9 was much more potent than scFv 17b, and more potent than or comparable to the best-characterized broadly neutralizing hmAbs IgG(1) b12, 2G12, 2F5 and 4E10. It also inhibited cell-to-cell transmission of HIV-1 with higher potency than enfuvirtide (T-20, Fuzeon). M9 competed with a sulfated CCR5 N-terminal peptide for binding to gp120-CD4 complex, suggesting an overlapping epitope with the coreceptor binding site. M9 did not react with phosphatidylserine (PS) and cardiolipin (CL), nor did it react with a panel of autoantigens in an antinuclear autoantibody (ANA) assay. We further found that escape mutants resistant to m9 did not emerge in an immune selection assay. These results suggest that m9 is a novel anti-HIV-1 candidate with potential therapeutic or prophylactic properties, and its epitope is a new target for drug or vaccine development.

Multivalent benzoboroxole functionalized polymers as gp120 glycan targeted microbicide entry inhibitors.

Microbicides are women-controlled prophylactics for sexually transmitted infections. The most important class of microbicides target HIV-1 and contain antiviral agents formulated for topical vaginal delivery. Identification of new viral entry inhibitors that target the HIV-1 envelope is important because they can inactivate HIV-1 in the vaginal lumen before virions can come in contact with CD4+ cells in the vaginal mucosa. Carbohydrate binding agents (CBAs) demonstrate the ability to act as entry inhibitors due to their ability to bind to glycans and prevent gp120 binding to CD4+ cells. However, as proteins they present significant challenges in regard to economical production and formulation for resource-poor environments. We have synthesized water-soluble polymer CBAs that contain multiple benzoboroxole moieties. A benzoboroxole-functionalized monomer was synthesized and incorporated into linear oligomers with 2-hydroxypropylmethacrylamide (HPMAm) at different feed ratios using free radical polymerization. The benzoboroxole small molecule analogue demonstrated weak affinity for HIV-1BaL gp120 by SPR; however, the 25 mol % functionalized benzoboroxole oligomer demonstrated a 10-fold decrease in the K(D) for gp120, suggesting an increased avidity for the multivalent polymer construct. High molecular weight polymers functionalized with 25, 50, and 75 mol % benzoboroxole were synthesized and tested for their ability to neutralize HIV-1 entry for two HIV-1 clades and both R5 and X4 coreceptor tropism. All three polymers demonstrated activity against all viral strains tested with EC(50)s that decrease from 15000 nM (1500 microg mL(-1)) for the 25 mol % functionalized polymers to 11 nM (1 microg mL(-1)) for the 75 mol % benzoboroxole-functionalized polymers. These polymers exhibited minimal cytotoxicity after 24 h exposure to a human vaginal cell line.

Safety, tolerability, and mechanisms of antiretroviral activity of pegylated interferon Alfa-2a in HIV-1-monoinfected participants: a phase II clinical trial.

BACKGROUND: To our knowledge, the antiviral activity of pegylated interferon alfa-2a has not been studied in participants with untreated human immunodeficiency virus type 1 (HIV-1) infection but without chronic hepatitis C virus (HCV) infection. METHODS: Untreated HIV-1-infected volunteers without HCV infection received 180 microg of pegylated interferon alfa-2a weekly for 12 weeks. Changes in plasma HIV-1 RNA load, CD4(+) T cell counts, pharmacokinetics, pharmacodynamic measurements of 2',5'-oligoadenylate synthetase (OAS) activity, and induction levels of interferon-inducible genes (IFIGs) were measured. Nonparametric statistical analysis was performed. RESULTS: Eleven participants completed 12 weeks of therapy. The median plasma viral load decrease and change in CD4(+) T cell counts at week 12 were 0.61 log(10) copies/mL (90% confidence interval [CI], 0.20-1.18 log(10) copies/mL) and -44 cells/microL (90% CI, -95 to 85 cells/microL), respectively. There was no correlation between plasma viral load decreases and concurrent pegylated interferon plasma concentrations. However, participants with larger increases in OAS level exhibited greater decreases in plasma viral load at weeks 1 and 2 (r = -0.75 [90% CI, -0.93 to -0.28] and r = -0.61 [90% CI, -0.87 to -0.09], respectively; estimated Spearman rank correlation). Participants with higher baseline IFIG levels had smaller week 12 decreases in plasma viral load (0.66 log(10) copies/mL [90% CI, 0.06-0.91 log(10) copies/mL]), whereas those with larger IFIG induction levels exhibited larger decreases in plasma viral load (-0.74 log(10) copies/mL [90% CI, -0.93 to -0.21 log(10) copies/mL]). CONCLUSION: Pegylated interferon alfa-2a was well tolerated and exhibited statistically significant anti-HIV-1 activity in HIV-1-monoinfected patients. The anti-HIV-1 effect correlated with OAS protein levels (weeks 1 and 2) and IFIG induction levels (week 12) but not with pegylated interferon concentrations.

Genetic signatures in the envelope glycoproteins of HIV-1 that associate with broadly neutralizing antibodies.

A steady increase in knowledge of the molecular and antigenic structure of the gp120 and gp41 HIV-1 envelope glycoproteins (Env) is yielding important new insights for vaccine design, but it has been difficult to translate this information to an immunogen that elicits broadly neutralizing antibodies. To help bridge this gap, we used phylogenetically corrected statistical methods to identify amino acid signature patterns in Envs derived from people who have made potently neutralizing antibodies, with the hypothesis that these Envs may share common features that would be useful for incorporation in a vaccine immunogen. Before attempting this, essentially as a control, we explored the utility of our computational methods for defining signatures of complex neutralization phenotypes by analyzing Env sequences from 251 clonal viruses that were differentially sensitive to neutralization by the well-characterized gp120-specific monoclonal antibody, b12. We identified ten b12-neutralization signatures, including seven either in the b12-binding surface of gp120 or in the V2 region of gp120 that have been previously shown to impact b12 sensitivity. A simple algorithm based on the b12 signature pattern was predictive of b12 sensitivity/resistance in an additional blinded panel of 57 viruses. Upon obtaining these reassuring outcomes, we went on to apply these same computational methods to define signature patterns in Env from HIV-1 infected individuals who had potent, broadly neutralizing responses. We analyzed a checkerboard-style neutralization dataset with sera from 69 HIV-1-infected individuals tested against a panel of 25 different Envs. Distinct clusters of sera with high and low neutralization potencies were identified. Six signature positions in Env sequences obtained from the 69 samples were found to be strongly associated with either the high or low potency responses. Five sites were in the CD4-induced coreceptor binding site of gp120, suggesting an important role for this region in the elicitation of broadly neutralizing antibody responses against HIV-1.

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

Growth hormone mitigates against lethal irradiation and enhances hematologic and immune recovery in mice and nonhuman primates.

Medications that can mitigate against radiation injury are limited. In this study, we investigated the ability of recombinant human growth hormone (rhGH) to mitigate against radiation injury in mice and nonhuman primates. BALB/c mice were irradiated with 7.5 Gy and treated post-irradiation with rhGH intravenously at a once daily dose of 20 microg/dose for 35 days. rhGH protected 17 out of 28 mice (60.7%) from lethal irradiation while only 3 out of 28 mice (10.7%) survived in the saline control group. A shorter course of 5 days of rhGH post-irradiation produced similar results. Compared with the saline control group, treatment with rhGH on irradiated BALB/c mice significantly accelerated overall hematopoietic recovery. Specifically, the recovery of total white cells, CD4 and CD8 T cell subsets, B cells, NK cells and especially platelets post radiation exposure were significantly accelerated in the rhGH-treated mice. Moreover, treatment with rhGH increased the frequency of hematopoietic stem/progenitor cells as measured by flow cytometry and colony forming unit assays in bone marrow harvested at day 14 after irradiation, suggesting the effects of rhGH are at the hematopoietic stem/progenitor level. rhGH mediated the hematopoietic effects primarily through their niches. Similar data with rhGH were also observed following 2 Gy sublethal irradiation of nonhuman primates. Our data demonstrate that rhGH promotes hematopoietic engraftment and immune recovery post the exposure of ionizing radiation and mitigates against the mortality from lethal irradiation even when administered after exposure.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

Immunization with cocktail of HIV-derived peptides in montanide ISA-51 is immunogenic, but causes sterile abscesses and unacceptable reactogenicity.

BACKGROUND: A peptide vaccine was produced containing B and T cell epitopes from the V3 and C4 Envelope domains of 4 subtype B HIV-1 isolates (MN, RF, CanO, & Ev91). The peptide mixture was formulated as an emulsion in incomplete Freund's adjuvant (IFA). METHODS: Low-risk, healthy adult subjects were enrolled in a randomized, placebo-controlled dose-escalation study, and selected using criteria specifying that 50% in each study group would be HLA-B7+. Immunizations were scheduled at 0, 1, and 6 months using a total peptide dose of 1 or 4 mg. Adaptive immune responses in16 vaccine recipients and two placebo recipients after the 2nd immunization were evaluated using neutralization assays of sera, as well as ELISpot and ICS assays of cryopreserved PBMCs to assess CD4 and CD8 T-cell responses. In addition, (51)Cr release assays were performed on fresh PBMCs following 14-day stimulation with individual vaccine peptide antigens. RESULTS: 24 subjects were enrolled; 18 completed 2 injections. The study was prematurely terminated because 4 vaccinees developed prolonged pain and sterile abscess formation at the injection site-2 after dose 1, and 2 after dose 2. Two other subjects experienced severe systemic reactions consisting of headache, chills, nausea, and myalgia. Both reactions occurred after the second 4 mg dose. The immunogenicity assessments showed that 6/8 vaccinees at each dose level had detectable MN-specific neutralizing (NT) activity, and 2/7 HLA-B7+ vaccinees had classical CD8 CTL activity detected. However, using both ELISpot and ICS, 8/16 vaccinees (5/7 HLA-B7+) and 0/2 controls had detectable vaccine-specific CD8 T-cell responses. Subjects with moderate or severe systemic or local reactions tended to have more frequent T cell responses and higher antibody responses than those with mild or no reactions. CONCLUSIONS: The severity of local responses related to the formulation of these four peptides in IFA is clinically unacceptable for continued development. Both HIV-specific antibody and T cell responses were induced and the magnitude of response correlated with the severity of local and systemic reactions. If potent adjuvants are necessary for subunit vaccines to induce broad and durable immune responses, careful, incremental clinical evaluation is warranted to minimize the risk of adverse events. TRIAL REGISTRATION: ClinicalTrials.gov NCT00000886.