Mucosal IL-4R antagonist HIV vaccination with SOSIP-gp140 booster can induce high-quality cytotoxic CD4+/CD8+ T cells and humoral responses in macaques.

Inducing humoral, cellular and mucosal immunity is likely to improve the effectiveness of HIV-1 vaccine strategies. Here, we tested a vaccine regimen in pigtail macaques using an intranasal (i.n.) recombinant Fowl Pox Virus (FPV)-gag pol env-IL-4R antagonist prime, intramuscular (i.m.) recombinant Modified Vaccinia Ankara Virus (MVA)-gag pol-IL-4R antagonist boost followed by an i.m SOSIP-gp140 boost. The viral vector-expressed IL-4R antagonist transiently inhibited IL-4/IL-13 signalling at the vaccination site. The SOSIP booster not only induced gp140-specific IgG, ADCC (antibody-dependent cellular cytotoxicity) and some neutralisation activity, but also bolstered the HIV-specific cellular and humoral responses. Specifically, superior sustained systemic and mucosal HIV Gag-specific poly-functional/cytotoxic CD4+ and CD8+ T cells were detected with the IL-4R antagonist adjuvanted strategy compared to the unadjuvanted control. In the systemic compartment elevated Granzyme K expression was linked to CD4+ T cells, whilst Granzyme B/TIA-1 to CD8+ T cells. In contrast, the cytotoxic marker expression by mucosal CD4+ and CD8+ T cells differed according to the mucosal compartment. This vector-based mucosal IL-4R antagonist/SOSIP booster strategy, which promotes cytotoxic mucosal CD4+ T cells at the first line of defence, and cytotoxic CD4+ and CD8+ T cells plus functional antibodies in the blood, may prove valuable in combating mucosal infection with HIV-1 and warrants further investigation.

Mucosal-associated invariant T (MAIT) cells are activated in the gastrointestinal tissue of patients with combination ipilimumab and nivolumab therapy-related colitis in a pathology distinct from ulcerative colitis.

The aim of this study was to investigate the pathogenesis of combination ipilimumab and nivolumab-associated colitis (IN-COL) by measuring gut-derived and peripheral blood mononuclear cell (GMNC; PBMC) profiles. We studied GMNC and PBMC from patients with IN-COL, IN-treated with no adverse-events (IN-NAE), ulcerative colitis (UC) and healthy volunteers using flow cytometry. In the gastrointestinal-derived cells we found high levels of activated CD8+ T cells and mucosal-associated invariant T (MAIT) cells in IN-COL, changes that were not evident in IN-NAE or UC. UC, but not IN-C, was associated with a high proportion of regulatory T cells (Treg ). We sought to determine if local tissue responses could be measured in peripheral blood. Peripherally, checkpoint inhibition instigated a rise in activated memory CD4+ and CD8+ T cells, regardless of colitis. Low circulating MAIT cells at baseline was associated with IN-COL patients compared with IN-NAE in one of two cohorts. UC, but not IN-COL, was associated with high levels of circulating plasmablasts. In summary, the alterations in T cell subsets measured in IN-COL-affected tissue, characterized by high levels of activated CD8+ T cells and MAIT cells and a low proportion of Treg , reflected a pathology distinct from UC. These tissue changes differed from the periphery, where T cell activation was a widespread on-treatment effect, and circulating MAIT cell count was low but not reliably predictive of colitis.

Comparison of the Freelite serum free light chain (SFLC) assay with serum and urine electrophoresis/immunofixation and the N Latex FLC assay.

Few reports have compared available serum free light chain (SFLC) assays. Here, a retrospective audit of the Freelite SFLC assay compared results to electrophoresis (EP)/immunofixation (IFX) and the N Latex FLC assay.A total of 244 samples collected over 3.5 months were studied using the Freelite and N Latex FLC nephelometry assays. Results were compared with serum and/or urine EP/IFX. The precision and linearity of the N Latex FLC assay was examined.Detectable paraprotein by serum or urine EP/IFX was present in 94% of samples with kappa and 100% with lambda FLC restriction. The correlation between the assays was higher for kappa (rho = 0.97) than lambda (rho = 0.89) especially when lambda results were above the upper limit of normal (rho = 0.62). Agreement in the categorical diagnosis as measured by the Cohen's kappa statistic was good (0.70). The N Latex FLC assay displayed good precision and linearity. In discordant samples the Freelite and N Latex FLC assays had equivalent agreement with IFX.Traditional methods of EP/IFX detected paraproteins in the majority of cases. Correlation between the Freelite and N Latex FLC assay is better for kappa than lambda FLC. The two assays are not entirely equivalent. Care should be taken by interpreting physicians and laboratories considering switching assays.

Anal intraepithelial neoplasia and squamous cell carcinoma in HIV-infected adults.

Anal cancer is one of the most common non-AIDS-defining malignancies in the era of combination antiretroviral therapy. Its precursor lesion, anal intraepithelial neoplasia (AIN), is highly prevalent in HIV-infected populations. More than 90% of anal squamous cell cancers are attributable to human papillomavirus (HPV). While the biology of HPV-related intraepithelial neoplasia is consistent across lower anogenital sites, the natural history of AIN is not well established and cannot be assumed to be identical to that of cervical intraepithelial neoplasia. Screening strategies to prevent anal cancer should be developed based on robust natural history data in HIV-infected and uninfected populations. Likewise, treatments need to be tested in randomized clinical trials, and reserved for those at significant risk of progression to cancer. This review covers the epidemiology, pathogenesis and immunology of HPV infection, AIN and anal cancer, and summarizes the current diagnosis, screening and treatment strategies in HIV-infected adults.

Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity.

Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells. We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process. We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.

Transmission of triple-class, drug-resistant HIV-1 in Australia.

We report the first two cases of transmitted triple-class, drug-resistant HIV-1 in Australia. Baseline testing of a newly diagnosed man showed four reverse transcriptase resistance mutations (affecting two drug classes) and six protease resistance mutations. A source patient was identified, and a likely second case newly infected 1 year later, suggesting sequential transmission. This raises therapeutic implications for the individual patients, as well as public health concerns.

Restoration of anti-tetanus toxoid responses in patients initiating highly active antiretroviral therapy with or without a boost immunization: an INITIO substudy.

INITIO is an open-labelled randomized trial evaluating first-line therapeutic strategies for human immunodeficiency virus-1 (HIV-1) infection. In an immunology substudy a tetanus toxoid booster (TTB) immunization was planned for 24 weeks after initiation of highly active antiretroviral therapy (HAART). All patients had received tetanus toxoid immunization in childhood. Generation of proliferative responses to tetanus toxoid was compared in two groups of patients, those receiving a protease inhibitor (PI)-sparing regimen (n = 21) and those receiving a PI-containing (n = 54) regimen. Fifty-two participants received a TTB immunization [PI-sparing (n = 15), PI-containing (n = 37)] and 23 participants did not [PI-sparing (n = 6) or PI-containing (n = 17)]. Cellular responses to tetanus antigen were monitored by lymphoproliferation at time of immunization and every 24 weeks to week 156. Proportions with a positive response (defined as stimulation index > or = 3 and Delta counts per minute > or = 3000) were compared at weeks 96 and 156. All analyses were intent-to-treat. Fifty-two participants had a TTB immunization at median 25 weeks; 23 patients did not. At weeks 96 and 156 there was no evidence of a difference in tetanus-specific responses, between those with or without TTB immunization (P = 0.2, P = 0.4). There was no difference in the proportion with response between those with PI-sparing or PI-containing regimens at both time-points (P = 0.8, P = 0.7). The proliferative response to tetanus toxoid was unaffected by initial HAART regimen. Anti-tetanus responses appear to reconstitute eventually in most patients over 156 weeks when treated successfully with HAART, irrespective of whether or not a TTB immunization has been administered.

Acutely dysregulated, chronically disabled by the enemy within: T-cell responses to HIV-1 infection.

Human immunodeficiency virus (HIV) infection causes chronic progressive immunodeficiency and immune dysregulaton. Although simple depletion of the major target of HIV infection, the CD4+ T cell, can explain much of the immunosuppression seen, there are multiple other factors contributing to the immune dysregulation. CD4+ T-cell depletion induces a range of homeostatic mechanisms that contribute to immune activation and cell turnover, providing a milieu conducive to further viral replication and cell destruction, resulting in functional defects in various lymphoid organs. These changes are progressive and in turn compromise the homeostatic processes. Further, the infection, like any other viral infection, provokes an active immune response consisting of both CD4+ and CD8+ T-cell responses. Both appear compromised, displaying aberrant memory cell production. While some of these defects result from viral variation and the chronicity of antigen presentation, other defects of memory cell production appear very early during the primary immune response limiting the viral specific T-cell responses from the outset. This, combined with the ability of the virus to escape any successful immune responses, results in an attenuated immune response that eventually becomes exhausted, characterized by progressive deficits in T-cell repertoire. Furthermore, negative regulatory mechanisms that normally control the immune response may be aberrantly invoked, perhaps directly by the virus, further compromising the efficacy of the immune response. Rational design of effective immunotherapies depends on a clear understanding of the processes compromising the immune response to HIV.

Influence of IFNgamma co-expression on the safety and antiviral efficacy of recombinant fowlpox virus HIV therapeutic vaccines following interruption of antiretroviral therapy.

Therapeutic immunization to stimulate host immune responses and control human immunodeficiency virus (HIV-1) replication is being investigated as a supplementary treatment for the management of HIV infection. On completion of an earlier study involving three vaccinations while taking combination antiretroviral therapy (CART), twenty-five subjects with plasma viral load (pVL) <50 copies/mL received a booster vaccination with either placebo (n = 7); fowl pox vaccine (rFPV) expressing HIV-1 Gag/Pol; [partial construct- PC (n = 8)] or rFPV coexpressing HIV-1 Gag/Pol and human interferon gamma[full construct - FC (n = 10)]. One week after the booster vaccination, participants stopped ART and were monitored for safety, pVL and immunological parameters for < or =20 weeks. The time weighted mean change (SD) from baseline plasma HIV RNA was 1.80 (0.72), 1.78 (0.91) and 0.96 (0.91) log(10) copies/mL for placebo, PC and FC recipients respectively (p = 0.06; mean differences between placebo and FC). Laboratory evaluations did not reveal differences in anti-HIV specific immune responses between study arms. No difference between treatment arms for host genetic factors known to affect pVL was demonstrated. In conclusion, vaccination with FC was associated with a trend toward lower rates of HIV replication following cessation of ART relative to placebo or PC. The promising antiretrovirological effect supports further study of FC in a larger trial with a broader population of patients with HIV disease.

Randomized, placebo-controlled, phase I/IIa evaluation of the safety and immunogenicity of fowlpox virus expressing HIV gag-pol and interferon-gamma in HIV-1 infected subjects.

We conducted a randomized, placebo-controlled double-blind trial to examine the safety and immunogenicity of a candidate HIV therapeutic vaccine based upon a recombinant fowl pox virus capable of coexpressing the human cytokine interferon-gamma and/or genes from HIV-1. Thirty-five eligible subjects were randomized (12 placebo, 11 fowlpox + HIV genes, 12 fowl pox + HIV genes + interferon gamma). All but one subject (placebo group) received three immunizations (by intramuscular injection on day 0, week 4 and week 12) and all completed 52 weeks of follow-up. All subjects continued to take combination antiretroviral therapy for the duration of study. There were no significant toxicity or safety concerns and the distribution of adverse events and their severity was consistent across each randomly assigned vaccine group. Comparison of placebo recipients with the combined recipients of the two vaccine constructs, in terms of anti-HIV gag ELISpot or lymphoproliferative responses, tended to favour the placebo group, but were not significantly different (difference in time-weighted mean change from baseline = 56 Spot forming units (sfu)/10(6) PBMC; p = 0.062 and 4.4 SI; p = 0.337). There were no significant changes in CTL responses by standard Cr(51) release assay. Anti-FPV antibodies were detected by week 14 in 0 placebo and 20 (87%) vaccine recipients. Although safe, neither vaccine construct appeared to possess detectable T-cell mediated anti-HIV immunogenic properties in HIV infected individuals, as measured by standard T cell assays.

Clustered mutations in HIV-1 gag are consistently required for escape from HLA-B27-restricted cytotoxic T lymphocyte responses.

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263-272, KRWIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Relative significance of different pathways of immune reconstitution in HIV type 1 infection as estimated by mathematical modeling.

A major goal of antiretroviral HIV-1 therapy is the reversal of HIV-1-associated immunological dysfunction. However, the pathogenetic mechanisms involved and their significance are largely unknown. On the basis of the life cycle of naive, activated, and memory CD4(+) T cell subsets, a mathematical model of immune reconstitution was developed and applied to data for T cell subsets in individuals with acute or chronic HIV-1 infection receiving antiretroviral therapy. The final model that most accurately fitted the data, and resulted in realistic estimates for CD4(+) T cell turnover, considered three pathways of immune reconstitution for naive cells, including thymic production, peripheral expansion, and redistribution of naive cells from lymphoid tissue. The reconstitution of the memory compartment was fitted through differentiation and expansion of naive cells and peripheral expansion of memory cells as well as redistribution of memory cells trapped in the lymphoid tissue. Estimated median half-lives for naive and memory CD4(+) T cells were 114 and 21 days, while total production rates were 9.1 x 10(7) and 2.4 x 10(9) cells/day, respectively. Peripheral expansion and thymic production contributed equally to the regeneration of naive cells, but peripheral expansion of memory cells was larger than production of these cells by differentiation of naive cells. A comparison of immune reconstitution in acute and chronic HIV-1 infection revealed that, after adjustment for age, the main difference was the more rapid release of a larger number of naive cells in treated acute HIV-1 infection. Thymic function and peripheral expansion rates, however, were similar in both cohorts.

Rapid restoration of CD4 T cell subsets in subjects receiving antiretroviral therapy during primary HIV-1 infection.

OBJECTIVE: To compare the effect of highly active antiretroviral therapy on immune reconstitution in subjects with acute and chronic HIV-1 infection. DESIGN: Prospective study including 58 treatment-naive subjects who commenced indinavir or nelfinavir and two nucleosides during primary (PHI; n = 28) or chronic HIV-1 infection (CHI; n = 30). METHODS: Naive (CD45RA+ 62L+), memory (CD45RA-) and activated (CD38+ HLA-DR+) T cell subsets were quantified at 1-2 monthly time intervals using 4-colour flow cytometry. RESULTS: At 1 year, HIV-1 RNA declined in both cohorts to undetectable levels (< 50 copies/ml), while median CD4 lymphocyte count increased from 470 to 758 x 10(6) cells/l in PHI and from 204 to 310 x 10(6) cells/l in CHI, reaching > 500 x 10(6) cells/l in 93% of PHI, but only in 37% of CHI subjects (P < 0.001). Naive CD4 lymphocytes increased from 106 to 176 x 10(6) cells/l in PHI and from 41 to 44 x 10(6) cells/l in CHI (PHI versus CHI at 12 months: P = 0.003), while memory cells rose from 368 to 573 x 10(6) cells/l in PHI and from 148 to 223 x 10(6) cells/l in CHI (P < 0.001). Early increases (< 3 months) of CD4 lymphocytes were larger in subjects with PHI, consisting of naive CD45RA+ CD62L+ as well as memory CD45RA- CD62L+ cells (P = 0.001). CD4 activation declined from 5 to 2% in PHI and from 13 to 6% in CHI (P = 0.001), while CD8 cell activation was reduced from 33 to 15% in PHI and from 42 to 19% in CHI (P = 0.02). CONCLUSION: Immune reconstitution was more complete, occurred earlier and comprised both naive and memory CD4 T lymphocytes in subjects who commenced antiretroviral therapy during primary HIV-1 infection.

Cytotoxic T lymphocyte responses to human immunodeficiency virus: control and escape.

Effective preventive and therapeutic intervention in individuals exposed to or infected with human immunodeficiency virus (HIV) depends, in part, on a clear understanding of the interactions between the virus and those elements of the host immune response which control viral replication. Recent advances have provided compelling evidence that cytotoxic T lymphocytes (CTLs) constitute an essential component of protective antiretroviral immunity. Here, we review briefly the significance of this work in the context of previous studies, and outline the mechanisms through which HIV evades CTL activity.

Functions of tetramer-stained HIV-specific CD4(+) and CD8(+) T cells.

Significant insight has been gained into constraints on the sensitivity and specificity of staining with class I tetramers. The function of the populations that are defined varies with the clinical situation. Insight has also been gained into the determinants of the CD8(+) T cell response during primary and chronic HIV infection. Human class II tetramers have been synthesised but their role in defining CD4(+) T cell function in HIV infection remains to be determined.

Early highly active antiretroviral therapy for acute HIV-1 infection preserves immune function of CD8+ and CD4+ T lymphocytes.

Highly active antiretroviral therapy (HAART) has been advocated for the management of primary HIV-1 infection without clear understanding of its immunological effects. Here, we demonstrate that early use of HAART during primary infection preserves HIV-specific CD8(+) T cells physically and functionally while HIV-specific T cell help is sustained. We also show that even transient administration of HAART at seroconversion can preserve HIV-specific immunity. In contrast, delayed initiation of HAART is associated with a progressive loss of HIV-specific CD8(+) T cells and absent HIV-specific T cell help. These results imply that HIV-specific T help is damaged during primary HIV-1 infection. Early drug treatment, which preserves this immunity, also preserves HIV-specific CD8(+) T cells. These results have implications for understanding the early pathogenesis of HIV-1 infection and suggest that acute HIV infection should be treated aggressively and as early as possible.

Potent antiretroviral therapy of primary human immunodeficiency virus type 1 (HIV-1) infection: partial normalization of T lymphocyte subsets and limited reduction of HIV-1 DNA despite clearance of plasma viremia.

Antiretroviral therapy commenced during primary human immunodeficiency virus type 1 (HIV-1) infection (PHI) may limit the extent of viral replication and prevent early loss of HIV-specific CD4 lymphocyte function. We studied the effect of current standard therapy (2 nucleoside analogues and a protease inhibitor) in 16 patients with symptomatic PHI. In the 13 patients who completed 1 year of treatment, plasma HIV RNA was <50 copies/mL and median CD4 cell counts were comparable to HIV-uninfected controls, with naive (CD45RA+CD62L+), primed (CD45RO+), and T cell receptor Vbeta subsets all within normal ranges. However, HIV-1 DNA levels in treated and untreated PHI patients were similar. Furthermore, CD8 cell counts remained elevated, including activated (CD38+HLA-DR+), replicating (Ki-67+), and cytotoxic (perforin+CD28-) lymphocytes. In conclusion, early antiretroviral therapy resulted in clearance of viremia and prevented loss of crucial CD4 subsets. The persistence of HIV-1 DNA together with increased CD8 T lymphocyte turnover and activation indicate continued expression of viral antigens.

Effect of protease therapy on cytokine secretion by peripheral blood mononuclear cells (PBMC) from HIV-infected subjects.

The viral load reduction seen in patients with late stage HIV infection treated with the protease inhibitor, ritonavir, is accompanied by increases in the in vitro proliferative responses generated by PBMC. The present study was undertaken to investigate which lymphocyte subsets generated these responses and the effects of therapy on cytokine production. Lymphoproliferation following phytohaemagglutinin (PHA) stimulation was studied by thymidine incorporation, and production of IL-2, interferon-gamma (IFN-gamma) and IL-4 was assessed by ELISA in 12 patients receiving ritonavir and seven receiving placebo in the context of randomized, blinded clinical trials. CD4+ cell-depleted and CD8+ cell-depleted subsets were obtained from PBMC by immunomagnetic bead depletion. At week 4 of therapy a two-fold or greater increase in proliferative responses was observed in 9/12 subjects receiving therapy, compared with 0/7 receiving placebo. Similarly there was a significant increase in IL-2 and IFN-gamma production of 2.7-fold (P = 0.02) and 1.7-fold (P = 0.03), respectively, in the treatment group compared with those receiving placebo. No change in IL-4 production was observed. Despite these increases, cytokine responses post-therapy were still reduced compared with both healthy controls and asymptomatic HIV-infected subjects. Increases in proliferative response and IL-2 production were greater in the CD8+ cell-depleted population than in the CD4+ cell-depleted population, whereas increases in IFN-gamma production were derived from the CD4+ cell-depleted population.

Therapeutic vaccination with p24-VLP and zidovudine augments HIV-specific cytotoxic T lymphocyte activity in asymptomatic HIV-infected individuals.

This study evaluates the impact of therapeutic vaccination with p24-VLP and zidovudine on the induction or maintenance of HIV-specific cytotoxic lymphocyte activity in a cohort of asymptomatic patients with CD4 counts greater than 400 cells/microl. In a dummy, randomized, phase II clinical trial of the therapeutic vaccine, participants were randomized to one of three arms for 6 months: p24-VLP (500 microg) in alum monthly plus zidovudine 200 mg tds, alum adjuvant plus zidovudine, or p24-VLP plus placebo. Subjects were studied for a total of 52 weeks from baseline. Monitoring included viral load, CD4 and CD8 counts, markers of immune activation, delayed-type hypersensitivity (DTH) skin testing, and cytotoxic T lymphocyte (CTL) measurement. The nine subjects who received p24-VLP and zidovudine had an augmentation and/or broadening of their CTL response compared with baseline (p = 0.004). The eight subjects receiving p24-VLP and seven subjects receiving zidovudine did not have a statistically significant increase or broadening of CTL activity. The augmentation of the CTL response in the subjects who received p24-VLP and zidovudine was not associated with a decline in viral load or an increase in CD8 counts. This study suggests that HIV-specific CTL activity can be augmented in HIV-infected individuals receiving p24-VLP and zidovudine, supporting the hypothesis of therapeutic vaccination in the presence of antiretroviral therapy.