Human cytomegalovirus-specific cytotoxic T cells. Relative frequency of stage-specific CTL recognizing the 72-kD immediate early protein and glycoprotein B expressed by recombinant vaccinia viruses.

CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium

Conditionally-live attenuated SIV upregulates global T effector memory cell frequency under replication permissive conditions.

Background: Live attenuated SIV induces potent protection against superinfection with virulent virus; however the mechanism of this vaccine effect is poorly understood. Such knowledge is important for the development of clinically acceptable vaccine modalities against HIV. Results: Using a novel, doxycycline dependent, replication-competent live-attenuated SIVmac239Δnef (SIVrtTAΔnef), we show that under replication-permissive conditions SIV-rtTAΔnef is fully viable. Twelve rhesus macaques were infected with a peak plasma vRNA on average two log10 lower than in 6 macaques infected with unconditionally replication-competent SIVΔnef. Consistent with the attenuated phenotype of the viruses the majority of animals displayed low or undetectable levels of viraemia by 42-84 days after infection. Next, comparison of circulating T cells before and after chronic infection with parental SIVΔnef revealed a profound global polarisation toward CD28-CCR7- T-effector memory 2 (TEM2) cells within CD95+CD4+ and CD95+CD8+ populations. Critically, a similar effect was seen in the CD95+ CD4+ population and to somewhat lesser extent in the CD95+ CD8+ population of SIV-rtTAΔnef chronically infected macaques that were maintained on doxycycline, but was not seen in animals from which doxycycline had been withdrawn. The proportions of gut-homing T-central memory (TCM) and TEM defined by the expression of α4β7 and CD95 and differential expression of CD28 were increased in CD4 and CD8 cells under replication competent conditions and gut-homing CD4 TCM were also significantly increased under non-permissive conditions. TEM2 polarisation was seen in the small intestines of animals under replication permissive conditions but the effect was less pronounced than in the circulation. Intracellular cytokine staining of circulating SIV-specific T cells for IL-2, IFN-γ, TNF-α and IL-17 showed that the extent of polyfunctionality in CD4 and CD8 T cells was associated with replication permissivity; however, signature patterns of cytokine combinations were not distinguishable between groups of macaques. Conclusion: Taken together our results show that the global T memory cell compartment is profoundly skewed towards a mature effector phenotype by attenuated SIV. Results with the replication-conditional mutant suggest that maintenance of this effect, that may be important in vaccine design, might require persistence of replicating virus

Complement-Mediated Virus Infectivity Neutralisation by HLA Antibodies Is Associated with Sterilising Immunity to SIV Challenge in the Macaque Model for HIV/AIDS.

Sterilising immunity is a desired outcome for vaccination against human immunodeficiency virus (HIV) and has been observed in the macaque model using inactivated simian immunodeficiency virus (SIV). This protection was attributed to antibodies specific for cell proteins including human leucocyte antigens (HLA) class I and II incorporated into virions during vaccine and challenge virus preparation. We show here, using HLA bead arrays, that vaccinated macaques protected from virus challenge had higher serum antibody reactivity compared with non-protected animals. Moreover, reactivity was shown to be directed against HLA framework determinants. Previous studies failed to correlate serum antibody mediated virus neutralisation with protection and were confounded by cytotoxic effects. Using a virus entry assay based on TZM-bl cells we now report that, in the presence of complement, serum antibody titres that neutralise virus infectivity were higher in protected animals. We propose that complement-augmented virus neutralisation is a key factor in inducing sterilising immunity and may be difficult to achieve with HIV/SIV Env-based vaccines. Understanding how to overcome the apparent block of inactivated SIV vaccines to elicit anti-envelope protein antibodies that effectively engage the complement system could enable novel anti-HIV antibody vaccines that induce potent, virolytic serological response to be developed

Phase I Randomised Clinical Trial of an HIV-1CN54, Clade C, Trimeric Envelope Vaccine Candidate Delivered Vaginally

We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18–45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 µg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women

Role of Occult and Post-acute Phase Replication in Protective Immunity Induced with a Novel Live Attenuated SIV Vaccine

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity

Early biodistribution and persistence of a protective live attenuated SIV vaccine elicits localised innate responses in multiple lymphoid tissues.

Vaccination of Mauritian cynomolgus macaques with the attenuated nef-truncated C8 variant of SIVmac251/32H (SIVmacC8) induces early, potent protection against pathogenic, heterologous challenge before the maturation of cognate immunity. To identify processes that contribute to early protection in this model the pathogenesis, anatomical distribution and viral vaccine kinetics were determined in relation to localised innate responses triggered by vaccination. The early biodistribution of SIVmacC8 was defined by rapid, widespread dissemination amongst multiple lymphoid tissues, detectable after 3 days. Cell-associated viral RNA dynamics identified mesenteric lymph nodes (MLN) and spleen, as well as the gut mucosae, as early major contributors of systemic virus burden. Rapid, localised infection was populated by discrete foci of persisting virus-infected cells. Localised productive infection triggered a broad innate response, with type-1 interferon sensitive IRF-7, STAT-1, TRIM5α and ApoBEC3G genes all upregulated during the acute phase but induction did not prevent viral persistence. Profound changes in vaccine-induced cell-surface markers of immune activation were detected on macrophages, B-cells and dendritic cells (DC-SIGN, S-100, CD40, CD11c, CD123 and CD86). Notably, high DC-SIGN and S100 staining for follicular and interdigitating DCs respectively, in MLN and spleen were detected by 3 days, persisting 20 weeks post-vaccination. Although not formally evaluated, the early biodistribution of SIVmacC8 simultaneously targets multiple lymphoid tissues to induce strong innate immune responses coincident at the same sites critical for early protection from wild-type viruses. HIV vaccines which stimulate appropriate innate, as well as adaptive responses, akin to those generated by live attenuated SIV vaccines, may prove the most efficacious

Immunity to HIV-1 Is Influenced by Continued Natural Exposure to Exogenous Virus

Unprotected sexual intercourse between individuals who are both infected with HIV-1 can lead to exposure to their partner's virus, and potentially to super-infection. However, the immunological consequences of continued exposure to HIV-1 by individuals already infected, has to our knowledge never been reported. We measured T cell responses in 49 HIV-1 infected individuals who were on antiretroviral therapy with suppressed viral loads. All the individuals were in a long-term sexual partnership with another HIV-1 infected individual, who was either also on HAART and suppressing their viral loads, or viremic (>9000 copies/ml). T cell responses to HIV-1 epitopes were measured directly ex-vivo by the IFN-γ enzyme linked immuno-spot assay and by cytokine flow cytometry. Sexual exposure data was generated from questionnaires given to both individuals within each partnership. Individuals who continued to have regular sexual contact with a HIV-1 infected viremic partner had significantly higher frequencies of HIV-1-specific T cell responses, compared to individuals with aviremic partners. Strikingly, the magnitude of the HIV-1-specific T cell response correlated strongly with the level and route of exposure. Responses consisted of both CD4+ and CD8+ T cell subsets. Longitudinally, decreases in exposure were mirrored by a lower T cell response. However, no evidence for systemic super-infection was found in any of the individuals. Continued sexual exposure to exogenous HIV-1 was associated with increased HIV-1-specific T cell responses, in the absence of systemic super-infection, and correlated with the level and type of exposure

Safety and Adherence to Intermittent Pre-Exposure Prophylaxis (PrEP) for HIV-1 in African Men Who Have Sex with Men and Female Sex Workers

Background Little is known about safety of and adherence to intermittent HIV PrEP regimens, which may be more feasible than daily dosing in some settings. We present safety and adherence data from the first trial of an intermittent PrEP regimen among Kenyan men who have sex with men (MSM) and female sex workers (FSW). Methods/Principal Findings MSM and FSW were randomized to daily oral FTC/TDF or placebo, or intermittent (Monday, Friday and within 2 hours after sex, not to exceed one dose per day) oral FTC/TDF or placebo in a 2:1:2:1 ratio; volunteers were followed monthly for 4 months. Adherence was assessed with the medication event monitoring system (MEMS). Sexual activity data were collected via daily text message (SMS) queries and timeline followback interviews with a onemonth recall period. Sixty-seven men and 5 women were randomized into the study. Safety was similar among all groups. Median MEMS adherence rates were 83% [IQR: 63–92] for daily dosing and 55% [IQR:28–78] for fixed intermittent dosing (p = 0.003), while adherence to any post-coital doses was 26% [IQR:14–50]. SMS response rates were low, which may have impaired measurement of post-coital dosing adherence. Acceptability of PrEP was high, regardless of dosing regimen. Conclusions/Significance Adherence to intermittent dosing regimens, fixed doses, and in particular coitally-dependent doses, may be more difficult than adherence to daily dosing. However, intermittent dosing may still be appropriate for PrEP if intracellular drug levels, which correlate with prevention of HIV acquisition, can be attained with less than daily dosing and if barriers to adherence can be addressed. Additional drug level data, qualitative data on adherence barriers, and better methods to measure sexual activity are necessary to determine whether adherence to post-coital PrEP could be comparable to more standard regimens</p

Neuropathology of wild-type and nef-attenuated T cell tropic simian immunodeficiency virus (SIVmac32H) and macrophage tropic neurovirulent SIVmac17E-Fr in cynomolgus macaques

The neuropathology of simian immunodeficiency (SIV) infection in cynomolgus macaques (Macaca fascicularis) was investigated following infection with either T cell tropic SIVmacJ5, SIVmacC8 or macrophage tropic SIVmac17E-Fr. Formalin fixed, paraffin embedded brain tissue sections were analysed using a combination of in situ techniques. Macaques infected with either wild-type SIVmacJ5 or neurovirulent SIVmac17E-Fr showed evidence of neuronal dephosphorylation, loss of oligodendrocyte and CCR5 staining, lack of microglial MHC II expression, infiltration by CD4+ and CD8+ T cells and mild astrocytosis. SIVmacJ5-infected animals exhibited activation of microglia whilst those infected with SIVmac17E-Fr demonstrated a loss of microglia staining. These results are suggestive of impaired central nervous system (CNS) physiology. Furthermore, infiltration by T cells into the brain parenchyma indicated disruption of the blood brain barrier (BBB). Animals infected with the Δnef-attenuated SIVmacC8 showed microglial activation and astrogliosis indicative of an inflammatory response, lack of MHC II and CCR5 staining and infiltration by CD8+ T cells. These results demonstrate that the SIV infection of cynomolgus macaque can be used as a model to replicate the range of CNS pathologies observed following HIV infection of humans and to investigate the pathogenesis of HIV associated neuropathology