Safety and immunogenicity of intramuscular, single-dose V590 (rVSV-SARS-CoV-2 Vaccine) in healthy adults: Results from a phase 1 randomised, double-blind, placebo-controlled, dose-ranging trial.

BACKGROUND: Vaccines against COVID-19 are needed to overcome challenges associated with mitigating the global pandemic. We report the safety and immunogenicity of V590, a live recombinant vesicular stomatitis virus-based COVID-19 vaccine candidate. METHODS: In this placebo-controlled, double-blind, three-part phase 1 study, healthy adults were randomised to receive a single intramuscular dose of vaccine or placebo. In Part 1, younger (18-54 years) and, in Part 2, older (≥55 years) adults seronegative for SARS-CoV-2 nucleocapsid received one of four V590 dose levels (5.00 × 105; 2.40 × 106; 1.15 × 107; or 5.55 × 107 plaque-forming units [pfu]) or placebo. In Part 3, a single V590 dose level (5.55 × 107 pfu) or placebo was administered to younger SARS-CoV-2 seropositive adults. Primary endpoints included adverse events (AEs) and for Parts 1 and 2 anti-SARS-CoV-2 serum neutralising antibody responses measured by 50% plaque reduction neutralisation (PRNT50) assay at Day 28. Registration NCT04569786 [P001-02]. FINDINGS: 232 participants were randomised and 219 completed the study. In seronegative participants, anti-SARS-CoV-2 spike-specific antibody responses to V590 were low and comparable to placebo across the lower dose levels. At the highest dose level (5.55 × 107 pfu), anti-SARS-CoV-2 spike-specific PRNT50 was 2.3-fold higher than placebo. The most frequently reported AEs were injection-site pain (38.4%), headache (15.1%) and fatigue (13.4%). INTERPRETATION: V590 was generally well-tolerated. However, Day 28 anti-SARS-Cov-2 spike-specific antibody responses in seronegative participants following a single intramuscular administration of V590 were not sufficient to warrant continued development. FUNDING: The study was funded by Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Acceptability and feasibility study of patient-specific 'tumouroids' as personalised treatment screening tools: Protocol for prospective tissue and data collection of participants with confirmed or suspected renal cell carcinoma.

INTRODUCTION: 'Personalised medicine' aims to tailor interventions to the individual, and has become one of the fastest growing areas of cancer research. One of these approaches is to harvest cancer cells from patients and grow them in the laboratory, which can then be subjected to treatments and the response assessed. We have developed a 3D tumour model with a complex protein matrix that mimics the tumour stroma, cell to cell and cell-matrix interactions seen in vivo, called a tumouroid. In this study, we test the acceptability and feasibility of using this model to establish patient-derived tumouroids. METHODS AND ANALYSIS: This is a first in-human study using prospective tissue and data collection of adult participants with confirmed or suspected renal cell carcinoma. The goals of the study are to assess patient acceptability to the use of patient-derived tumour models for future treatment decisions, and to assess the feasibility of generating patient-specific renal cancer tumouroids that can be challenged with drugs. These goals will be realised through the collection of tumour samples (expected n = 10), participant-completed questionnaires (expected n = 10), and in-depth semi-structured interviews with patients (expected n = 5). Collected multiregional tumour samples will be dissociated to isolate primary cells which are then expanded in vitro and incorporated into tumouroids. Drug challenge will ensue and the response will be categorised into "responder", "weak responder", and "non-responder". Statistical analysis will be descriptive. ETHICS AND DISSEMINATION: The study has ethical approval (REC reference 17/LO/1744). Findings will be made available to patients, clinicians, funders, and the National Health Service (NHS) through presentations at national and international meetings, peer-reviewed publications, social media and patient support groups. TRIAL REGISTRATION: Registered on ClinicalTrials.gov (NCT03300102).

Envelope-Specific Recognition Patterns of HIV Vaccine-Induced IgG Antibodies Are Linked to Immunogen Structure and Sequence.

Background: A better understanding of the parameters influencing vaccine-induced IgG recognition of individual antigenic regions and their variants within the HIV Envelope protein (Env) can help to improve design of preventive HIV vaccines. Methods: Env-specific IgG responses were mapped in samples of the UKHVC003 Standard Group (UK003SG, n = 11 from UK) and TaMoVac01 (TMV01, n = 17 from Tanzania) HIV vaccine trials. Both trials consisted of three immunizations with DNA, followed by two boosts with recombinant Modified Vaccinia Virus Ankara (MVA), either mediating secretion of gp120 (UK003SG) or the presentation of cell membrane bound gp150 envelopes (TMV01) from infected cells, and an additional two boosts with 5 µg of CN54gp140 protein adjuvanted with glucopyranosyl lipid adjuvant (GLA). Env immunogen sequences in UK003SG were solely based on the clade C isolate CN54, whereas in TMV01 these were based on clades A, C, B, and CRF01AE. The peptide microarray included 8 globally representative Env sequences, CN54gp140 and the MVA-encoded Env immunogens from both trials, as well as additional peptide variants for hot spots of immune recognition. Results: After the second MVA boost, UK003SG vaccinees almost exclusively targeted linear, non-glycosylated antigenic regions located in the inter-gp120 interface. In contrast, TMV01 recipients most strongly targeted the V2 region and an immunodominant region in gp41. The V3 region was frequently targeted in both trials, with a higher recognition magnitude for diverse antigenic variants observed in the UK003SG (p < 0.0001). After boosting with CN54gp140/GLA, the overall response magnitude increased with a more comparable recognition pattern of antigenic regions and variants between the two trials. Recognition of most immunodominant regions within gp120 remained significantly stronger in UK003SG, whereas V2-region recognition was not boosted in either group. Conclusions: IgG recognition of linear antigenic Env regions differed between the two trials particularly after the second MVA boost. Structural features of the MVA-encoded immunogens, such as secreted, monomeric gp120 vs. membrane-anchored, functional gp150, and differences in prime-boost immunogen sequence variability most probably contributed to these differences. Prime-boosting with multivalent Env immunogens during TMV01 did not improve variant cross-recognition of immunodominant peptide variants in the V3 region.

The Safety and Immunogenicity of GTU®MultiHIV DNA Vaccine Delivered by Transcutaneous and Intramuscular Injection With or Without Electroporation in HIV-1 Positive Subjects on Suppressive ART.

Previous studies have shown targeting different tissues via the transcutaneous (TC) and intramuscular injection (IM) with or without electroporation (EP) has the potential to trigger immune responses to DNA vaccination. The CUTHIVTHER 001 Phase I/II randomized controlled clinical trial was designed to determine whether the mode of DNA vaccination delivery (TC+IM or EP+IM) could influence the quality and function of induced cellular immune responses compared to placebo, in an HIV positive clade B cohort on antiretroviral therapy (ART). The GTU®MultiHIV B DNA vaccine DNA vaccine encoded a MultiHIV B clade fusion protein to target the cellular response. Overall the vaccine and regimens were safe and well-tolerated. There were robust pre-vaccination IFN-γ responses with no measurable change following vaccination compared to placebo. However, modest intracellular cytokine staining (ICS) responses were seen in the TC+IM group. A high proportion of individuals demonstrated potent viral inhibition at baseline that was not improved by vaccination. These results show that HIV positive subjects with nadir CD4+ counts ≥250 on suppressive ART display potent levels of cellular immunity and viral inhibition, and that DNA vaccination alone is insufficient to improve such responses. These data suggest that more potent prime-boost vaccination strategies are likely needed to improve pre-existing responses in similar HIV-1 cohorts (This study has been registered at http://ClinicalTrials.gov under registration no. NCT02457689).

Combined Skin and Muscle DNA Priming Provides Enhanced Humoral Responses to a Human Immunodeficency Virus Type 1 Clade C Envelope Vaccine.

Intradermal (i.d.) and intramuscular (i.m.) injections when administered with or without electroporation (EP) have the potential to tailor the immune response to DNA vaccination. This Phase I randomized controlled clinical trial in human immunodeficiency virus type 1-negative volunteers investigated whether the site and mode of DNA vaccination influences the quality of induced cellular and humoral immune responses following the DNA priming phase and subsequent protein boost with recombinant clade C CN54 gp140. A strategy of concurrent i.d. and i.m. DNA immunizations administered with or without EP was adopted. Subtle differences were observed in the shaping of vaccine-induced virus-specific CD4+ and CD8+ T cell-mediated immune responses between groups receiving: i.d.EP + i.m., i.d. + i.m.EP, and i.d.EP + i.m.EP regimens. The DNA priming phase induced 100% seroconversion in all of the groups. A single, non-adjuvanted protein boost induced a rapid and profound increase in binding antibodies in all groups, with a trend for higher responses in i.d.EP + i.m.EP. The magnitude of antigen-specific binding immunoglobulin G correlated with neutralization of closely matched clade C 93MW965 virus and Fc-dimer receptor binding (FcγRIIa and FcγRIIIa). These results offer new perspectives on the use of combined skin and muscle DNA immunization in priming humoral and cellular responses to recombinant protein.

Nasal IgA Provides Protection against Human Influenza Challenge in Volunteers with Low Serum Influenza Antibody Titre.

In spite of there being a number of vaccines, influenza remains a significant global cause of morbidity and mortality. Understanding more about natural and vaccine induced immune protection against influenza infection would help to develop better vaccines. Virus specific IgG is a known correlate of protection, but other factors may help to reduce viral load or disease severity, for example IgA. In the current study we measured influenza specific responses in a controlled human infection model using influenza A/California/2009 (H1N1) as the challenge agent. Volunteers were pre-selected with low haemagglutination inhibition (HAI) titres in order to ensure a higher proportion of infection; this allowed us to explore the role of other immune correlates. In spite of HAI being uniformly low, there were variable levels of H1N1 specific IgG and IgA prior to infection. There was also a range of disease severity in volunteers allowing us to compare whether differences in systemic and local H1N1 specific IgG and IgA prior to infection affected disease outcome. H1N1 specific IgG level before challenge did not correlate with protection, probably due to the pre-screening for individuals with low HAI. However, the length of time infectious virus was recovered from the nose was reduced in patients with higher pre-existing H1N1 influenza specific nasal IgA or serum IgA. Therefore, IgA contributes to protection against influenza and should be targeted in vaccines.

Comparison of mucosal lining fluid sampling methods and influenza-specific IgA detection assays for use in human studies of influenza immunity.

We need greater understanding of the mechanisms underlying protection against influenza virus to develop more effective vaccines. To do this, we need better, more reproducible methods of sampling the nasal mucosa. The aim of the current study was to compare levels of influenza virus A subtype-specific IgA collected using three different methods of nasal sampling. Samples were collected from healthy adult volunteers before and after LAIV immunization by nasal wash, flocked swabs and Synthetic Absorptive Matrix (SAM) strips. Influenza A virus subtype-specific IgA levels were measured by haemagglutinin binding ELISA or haemagglutinin binding microarray and the functional response was assessed by microneutralization. Nasosorption using SAM strips lead to the recovery of a more concentrated sample of material, with a significantly higher level of total and influenza H1-specific IgA. However, an equivalent percentage of specific IgA was observed with all sampling methods when normalized to the total IgA. Responses measured using a recently developed antibody microarray platform, which allows evaluation of binding to multiple influenza strains simultaneously with small sample volumes, were compared to ELISA. There was a good correlation between ELISA and microarray values. Material recovered from SAM strips was weakly neutralizing when used in an in vitro assay, with a modest correlation between the level of IgA measured by ELISA and neutralization, but a greater correlation between microarray-measured IgA and neutralizing activity. In conclusion we have tested three different methods of nasal sampling and show that flocked swabs and novel SAM strips are appropriate alternatives to traditional nasal washes for assessment of mucosal influenza humoral immunity.

A Comparative Phase I Study of Combination, Homologous Subtype-C DNA, MVA, and Env gp140 Protein/Adjuvant HIV Vaccines in Two Immunization Regimes.

There remains an urgent need for a prophylactic HIV vaccine. We compared combined MVA and adjuvanted gp140 to sequential MVA/gp140 after DNA priming. We expected Env-specific CD4+ T-cells after DNA and MVA priming, and Env-binding antibodies in 100% individuals after boosting with gp140 and that combined vaccines would not compromise safety and might augment immunogenicity. Forty volunteers were primed three times with DNA plasmids encoding (CN54) env and (ZM96) gag-pol-nef at 0, 4 and 8 weeks then boosted with MVA-C (CN54 env and gag-pol-nef) and glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) adjuvanted CN54gp140. They were randomised to receive them in combination at the same visit at 16 and 20 weeks (accelerated) or sequentially with MVA-C at 16, 20, and GLA-AF/gp140 at 24 and 28 weeks (standard). All vaccinations were intramuscular. Primary outcomes included ≥grade 3 safety events and the titer of CN54gp140-specific binding IgG. Other outcomes included neutralization, binding antibody specificity and T-cell responses. Two participants experienced asymptomatic ≥grade 3 transaminitis leading to discontinuation of vaccinations, and three had grade 3 solicited local or systemic reactions. A total of 100% made anti-CN54gp140 IgG and combining vaccines did not significantly alter the response; geometric mean titer 6424 (accelerated) and 6578 (standard); neutralization of MW965.2 Tier 1 pseudovirus was superior in the standard group (82 versus 45% responders, p = 0.04). T-cell ELISpot responses were CD4+ and Env-dominant; 85 and 82% responding in the accelerated and standard groups, respectively. Vaccine-induced IgG responses targeted multiple regions within gp120 with the V3 region most immunodominant and no differences between groups detected. Combining MVA and gp140 vaccines did not result in increased adverse events and did not significantly impact upon the titer of Env-specific binding antibodies, which were seen in 100% individuals. The approach did however affect other immune responses; neutralizing antibody responses, seen only to Tier 1 pseudoviruses, were poorer when the vaccines were combined and while T-cell responses were seen in >80% individuals in both groups and similarly CD4 and Env dominant, their breadth/polyfunctionality tended to be lower when the vaccines were combined, suggesting attenuation of immunogenicity and cautioning against this accelerated regimen.

Comparative Immunogenicity of HIV-1 gp140 Vaccine Delivered by Parenteral, and Mucosal Routes in Female Volunteers; MUCOVAC2, A Randomized Two Centre Study.

BACKGROUND: Defining optimal routes for induction of mucosal immunity represents an important research priority for the HIV-1 vaccine field. In particular, it remains unclear whether mucosal routes of immunization can improve mucosal immune responses. METHODS: In this randomized two center phase I clinical trial we evaluated the systemic and mucosal immune response to a candidate HIV-1 Clade C CN54gp140 envelope glycoprotein vaccine administered by intramuscular (IM), intranasal (IN) and intravaginal (IVAG) routes of administration in HIV negative female volunteers. IM immunizations were co-administered with Glucopyranosyl Lipid Adjuvant (GLA), IN immunizations with 0.5% chitosan and IVAG immunizations were administered in an aqueous gel. RESULTS: Three IM immunizations of CN54 gp140 at either 20 or 100 µg elicited significantly greater systemic and mucosal antibodies than either IN or IVAG immunizations. Following additional intramuscular boosting we observed an anamnestic antibody response in nasally primed subjects. Modest neutralizing responses were detected against closely matched tier 1 clade C virus in the IM groups. Interestingly, the strongest CD4 T-cell responses were detected after IN and not IM immunization. CONCLUSIONS: These data show that parenteral immunization elicits systemic and mucosal antibodies in women. Interestingly IN immunization was an effective prime for IM boost, while IVAG administration had no detectable impact on systemic or mucosal responses despite IM priming. CLINICAL TRIALS REGISTRATION: EudraCT 2010-019103-27 and the UK Clinical Research Network (UKCRN) Number 11679.

Superior Efficacy of a Human Immunodeficiency Virus Vaccine Combined with Antiretroviral Prevention in Simian-Human Immunodeficiency Virus-Challenged Nonhuman Primates.

UNLABELLED: Although vaccines and antiretroviral (ARV) prevention have demonstrated partial success against human immunodeficiency virus (HIV) infection in clinical trials, their combined introduction could provide more potent protection. Furthermore, combination approaches could ameliorate the potential increased risk of infection following vaccination in the absence of protective immunity. We used a nonhuman primate model to determine potential interactions of combining a partially effective ARV microbicide with an envelope-based vaccine. The vaccine alone provided no protection from infection following 12 consecutive low-dose intravaginal challenges with simian-HIV strain SF162P3, with more animals infected compared to naive controls. The microbicide alone provided a 68% reduction in the risk of infection relative to that of the vaccine group and a 45% reduction relative to that of naive controls. The vaccine-microbicide combination provided an 88% reduction in the per-exposure risk of infection relative to the vaccine alone and a 79% reduction relative to that of the controls. Protected animals in the vaccine-microbicide group were challenged a further 12 times in the absence of microbicide and demonstrated a 98% reduction in the risk of infection. A total risk reduction of 91% was observed in this group over 24 exposures (P = 0.004). These important findings suggest that combined implementation of new biomedical prevention strategies may provide significant gains in HIV prevention. IMPORTANCE: There is a pressing need to maximize the impact of new biomedical prevention tools in the face of the 2 million HIV infections that occur each year. Combined implementation of complementary biomedical approaches could create additive or synergistic effects that drive improved reduction of HIV incidence. Therefore, we assessed a combination of an untested vaccine with an ARV-based microbicide in a nonhuman primate vaginal challenge model. The vaccine alone provided no protection (and may have increased susceptibility to a simian-HIV vaginal challenge), while the microbicide reduced the infection risk compared to that of vaccinated and naive animals. Importantly, the combined interventions provided the greatest level of protection, which was sustained following withdrawal of the microbicide. The data suggest that provision of ARV prophylaxis during vaccination reduces the potential for unexpected increased risks of infection following immunization and augments vaccine efficacy. These findings are important for the potential adoption of ARV prophylaxis as the baseline intervention for future HIV/AIDS vaccines.

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates.

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.

Enhanced immunogenicity of an HIV-1 DNA vaccine delivered with electroporation via combined intramuscular and intradermal routes.

UNLABELLED: It is accepted that an effective prophylactic HIV-1 vaccine is likely to have the greatest impact on viral transmission rates. As previous reports have implicated DNA-priming, protein boost regimens to be efficient activators of humoral responses, we sought to optimize this regimen to further augment vaccine immunogenicity. Here we evaluated single versus concurrent intradermal (i.d.) and intramuscular (i.m.) vaccinations as a DNA-priming strategy for their abilities to elicit humoral and cellular responses against a model HIV-1 vaccine antigen, CN54-gp140. To further augment vaccine-elicited T and B cell responses, we enhanced cellular transfection with electroporation and then boosted the DNA-primed responses with homologous protein delivered subcutaneously (s.c.), intranasally (i.n.), i.m., or transcutaneously (t.c.). In mice, the concurrent priming regimen resulted in significantly elevated gamma interferon T cell responses and high-avidity antigen-specific IgG B cell responses, a hallmark of B cell maturation. Protein boosting of the concurrent DNA strategy further enhanced IgG concentrations but had little impact on T cell reactivity. Interestingly protein boosting by the subcutaneous route increased antibody avidity to a greater extent than protein boosting by either the i.m., i.n., or t.c. route, suggesting that this route may be preferential for driving B cell maturation. Using an alternative and larger animal model, the rabbit, we found the concurrent DNA-priming strategy followed by s.c. protein boosting to again be capable of eliciting high-avidity humoral responses and to also be able to neutralize HIV-1 pseudoviruses from diverse clades (clades A, B, and C). Taken together, we show that concurrent multiple-route DNA vaccinations induce strong cellular immunity, in addition to potent and high-avidity humoral immune responses. IMPORTANCE: The route of vaccination has profound effects on prevailing immune responses. Due to the insufficient immunogenicity and protection of current DNA delivery strategies, we evaluated concurrent DNA delivery via simultaneous administration of plasmid DNA by the i.m. and i.d. routes. The rationale behind this study was to provide clear evidence of the utility of concurrent vaccinations for an upcoming human clinical trial. Furthermore, this work will guide future preclinical studies by evaluating the use of model antigens and plasmids for prime-boost strategies. This paper will be of interest not only to virologists and vaccinologists working in the HIV field but also to researchers working in other viral vaccine settings and, critically, to the wider field of vaccine delivery.

Glucopyranosyl lipid A adjuvant significantly enhances HIV specific T and B cell responses elicited by a DNA-MVA-protein vaccine regimen.

Using a unique vaccine antigen matched and single HIV Clade C approach we have assessed the immunogenicity of a DNA-poxvirus-protein strategy in mice and rabbits, administering MVA and protein immunizations either sequentially or simultaneously and in the presence of a novel TLR4 adjuvant, GLA-AF. Mice were vaccinated with combinations of HIV env/gag-pol-nef plasmid DNA followed by MVA-C (HIV env/gag-pol-nef) with HIV CN54gp140 protein (+/-GLA-AF adjuvant) and either co-administered in different muscles of the same animal with MVA-C or given sequentially at 3-week intervals. The DNA prime established a population of B cells that were able to mount a statistically significant anamnestic response to the boost vaccines. The greatest antigen-specific antibody response was observed in animals that received all vaccine components. Moreover, a high proportion of the total mucosal IgG (20 - 50%) present in the vaginal vault of these vaccinated animals was vaccine antigen-specific. The potent elicitation of antigen-specific immune responses to this vaccine modality was also confirmed in rabbits. Importantly, co-administration of MVA-C with the GLA-AF adjuvanted HIV CN54gp140 protein significantly augmented the antigen-specific T cell responses to the Gag antigen, a transgene product expressed by the MVA-C vector in a separate quadriceps muscle. We have demonstrated that co-administration of MVA and GLA-AF adjuvanted HIV CN54gp140 protein was equally effective in the generation of humoral responses as a sequential vaccination modality thus shortening and simplifying the immunization schedule. In addition, a significant further benefit of the condensed vaccination regime was that T cell responses to proteins expressed by the MVA-C were potently enhanced, an effect that was likely due to enhanced immunostimulation in the presence of systemic GLA-AF.

Phase I randomised clinical trial of an HIV-1(CN54), clade C, trimeric envelope vaccine candidate delivered vaginally.

UNLABELLED: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.

Antibody responses after intravaginal immunisation with trimeric HIV-1 CN54 clade C gp140 in Carbopol gel are augmented by systemic priming or boosting with an adjuvanted formulation.

Optimum strategies to elicit and maintain antibodies at mucosal portals of virus entry are critical for the development of vaccines against human immunodeficiency virus (HIV). Here we show in non-human primates that a novel regimen of repeated intravaginal delivery of a non-adjuvanted, soluble recombinant trimeric HIV-1(CN54) clade C envelope glycoprotein (gp140) administered in Carbopol gel can prime for B-cell responses even in the absence of seroconversion. Following 3 cycles of repeated intravaginal administration, throughout each intermenses interval, 3 of 4 macaques produced or boosted systemic and mucosally-detected antibodies upon intramuscular immunisation with gp140 formulated in AS01 adjuvant. Reciprocally, a single intramuscular immunisation primed 3 of 4 macaques for antibody boosting after a single cycle of intravaginal immunisation. Virus neutralising activity was detected against clade C and clade B HIV-1 envelopes but was restricted to highly neutralisation sensitive pseudoviruses.

Prevention of SIV rectal transmission and priming of T cell responses in macaques after local pre-exposure application of tenofovir gel.

BACKGROUND: The rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model. METHODS AND FINDINGS: A total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-gamma)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/10(6) PBMC to 261 spot forming cells (SFC)/10(6) PBMC. CONCLUSIONS: These results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.

Modelling cytomegalovirus replication patterns in the human host: factors important for pathogenesis.

Human cytomegalovirus can cause a diverse range of diseases in different immunocompromised hosts. The pathogenic mechanisms underlying these diseases have not been fully elucidated, though the maximal viral load during infection is strongly correlated with the disease. However, concentrating on single viral load measures during infection ignores valuable information contained during the entire replication history up to the onset of disease. We use a statistical model that allows all viral load data sampled during infection to be analysed, and have applied it to four immunocompromised groups exhibiting five distinct cytomegalovirus-related diseases. The results show that for all diseases, peaks in viral load contribute less to disease progression than phases of low virus load with equal amount of viral turnover. The model accurately predicted the time of disease onset for fever, gastrointestinal disease and pneumonitis but not for hepatitis and retinitis, implying that other factors may be involved in the pathology of these diseases.

Macaques infected long-term with attenuated simian immunodeficiency virus (SIVmac) remain resistant to wild-type challenge, despite declining cytotoxic T lymphocyte responses to an immunodominant epitope.

To further investigate mechanisms of protective immunity that are induced by live, attenuated simian immunodeficiency virus (SIV), three macaques were infected with SIVmacGX2, a nef-disrupted molecular clone. In two of these animals, which expressed the MamuA*01 major histocompatibility complex class I allele, loss of functional activity against an SIV-Gag-encoded immunodominant cytotoxic T lymphocyte (CTL) epitope was observed following prolonged infection. Nonetheless, all three animals were resistant to challenge with an uncloned pool of wild-type SIVmac, whereas four naïve controls became infected. Tetramer staining revealed the rapid generation of CD8+ T-cell responses against gag- and tat-encoded immunodominant epitopes in MamuA*01+ challenge controls. The dynamics of these T-cell responses to the wild-type virus were similar to those observed following primary infection of the vaccine group with attenuated virus. In contrast, neither tetramer staining nor gamma interferon ELISpot assay revealed an immediate, systemic, anamnestic response in the wild-type-challenged, attenuated SIV-infected animals. Functional CTL capacity had not been lost in this group, as lytic activity was still evident 17 weeks after challenge. Both attenuated and wild-type viruses induced a disseminated CD8+ T-cell response, which was of a higher magnitude in lymphoid tissues than in the periphery. These results suggest that, at least as measured in the periphery, protection against wild-type infection that is induced by live, attenuated SIV is not dependent on a rechallenge-driven expansion of immunodominant epitope-specific CD8+ T cells and, therefore, pre-existing activity may be sufficient to prevent superinfection.

Prospective study of the effects of antiretroviral therapy on Kaposi sarcoma--associated herpesvirus infection in patients with and without Kaposi sarcoma.

OBJECTIVE: To investigate the effect of highly active antiretroviral therapy (HAART) on circulating Kaposi sarcoma (KS)-associated herpesvirus (KSHV) load in HIV-infected individuals with and without KS. DESIGN: Twenty-nine HIV-infected subjects (21 with KS and 8 without KS) were recruited for the study; they were prospectively studied before and at regular intervals during HAART. METHODS: Patients with KS were clinically assessed using Adult Clinical Trials Group (ACTG) criteria, and sequential blood samples were obtained from all patients for determination of plasma HIV-1 load, CD4 cell count, titer of antibody to KSHV, and KSHV load. RESULTS: Ten of 21 patients with KS had a favorable KS response (complete response, 6; partial response, 4) with HAART alone. Of the 20 subjects with detectable KSHV viremia prior to HAART, over one half (12 [60%]) had an undetectable KSHV load with antiretroviral therapy. There was no significant difference between subjects receiving protease inhibitor- or nonnucleoside reverse transcriptase inhibitor-based treatment combinations. Achieving undetectable KSHV viremia is associated with a better clinical outcome in patients with KS. CONCLUSIONS: To our knowledge, we demonstrate for the first time that both protease inhibitor-based and nonnucleoside reverse transcriptase inhibitor-based antiretroviral treatment combinations may lead to an undetectable KSHV load and confirm that an undetectable KSHV load is associated with KS regression. There was no clear association between CD4 cell count response and KS response to HAART, but there was a significant relationship between HIV load response to HAART and clinical improvement of KS.

Identification of Kaposi's sarcoma-associated herpesvirus (KSHV)-specific cytotoxic T-lymphocyte epitopes and evaluation of reconstitution of KSHV-specific responses in human immunodeficiency virus type 1-Infected patients receiving highly active antiretroviral therapy.

Following the introduction of highly active antiretroviral therapy (HAART), the incidence of Kaposi's sarcoma (KS) has significantly declined in human immunodeficiency virus type 1 (HIV-1)-positive (HIV-1(+)) individuals and clinical remission is often observed. We hypothesize that these effects are partly due to anti-KS-associated herpesvirus (KSHV) immune restoration. Here, 15-mer overlapping peptides from proteins K12 and K8.1 were used to identify novel KSHV-specific cytotoxic T-lymphocyte epitopes. Three immunogenic peptides, two lytic and one latent, were subsequently used to monitor the anti-KSHV CD8(+) T-cell responses in a cohort of 19 HIV-1(+) KSHV(+/-) KS(+/-) individuals during 52 weeks of HAART. KSHV and HIV-1 loads, KSHV antibody titers, and both CD4(+) and CD8(+) T-lymphocyte counts were enumerated. Prior to HAART, the total number of spot-forming cells (SFC) for all three peptides correlated with both CD4(+) and CD8(+) T-lymphocyte counts (P < or = 0.05) in the KSHV-positive KS-positive cohort (n = 11). Following 52 weeks of HAART, significant decreases in HIV-1 and KSHV loads were associated with significant increases in CD4(+) T-lymphocyte counts and number of SFC for the three KSHV-specific peptides. Although these increases were modest in comparison to the number of SFC observed with the HIV-1 gag peptide SLYNTVATL, they represented a fourfold increase from the baseline, continuing an upward trend to week 52.