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One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed "prime-pull", has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the "pull" stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of "prime-pull" has only a modest effect on B cells and antibody.

Neutralizing antibodies may have critical importance in immunity against human immunodeficiency virus type 1 (HIV-1) infection. However, the amount of protective antibody needed at mucosal surfaces has not been fully established. Here, we evaluated systemic and mucosal pharmacokinetics (PK) and pharmacodynamics (PD) of 2F5 IgG and 2F5 Fab fragments with respect to protection against vaginal challenge with simian-human immunodeficiency virus-BaL in macaques. Antibody assessment demonstrated that 2F5 IgG was more potent than polymeric forms (IgM and IgA) across a range of cellular and tissue models. Vaginal challenge studies demonstrated a dose-dependent protection for 2F5 IgG and no protection with 2F5 Fab despite higher vaginal Fab levels at the time of challenge. Animals receiving 50 or 25 mg/kg of body weight 2F5 IgG were completely protected, while 3/5 animals receiving 5 mg/kg were protected. In the control animals, infection was established by a minimum of 1 to 4 transmitted/founder (T/F) variants, similar to natural human infection by this mucosal route; in the two infected animals that had received 5 mg 2F5 IgG, infection was established by a single T/F variant. Serum levels of 2F5 IgG were more predictive of sterilizing protection than measured vaginal levels. Fc-mediated antiviral activity did not appear to influence infection of primary target cells in cervical explants. However, PK studies highlighted the importance of the Fc portion in tissue biodistribution. Data presented in this study may be important in modeling serum levels of neutralizing antibodies that need to be achieved by either vaccination or passive infusion to prevent mucosal acquisition of HIV-1 infection in humans.

Human immunodeficiency virus type 1 (HIV-1) transmission results from infection with one or a small number of variants from the donor quasispecies. Transmitted/founder (T/F) viruses have recently been identified from acutely infected patients, but the way in which they interact with primary targets of HIV-1 infection is poorly understood. We have conducted a biological characterization of a panel of subtype B T/F acute and chronic envelope (Env)-expressing chimeric virus in primary human target cells and mucosal tissues. Both acute and chronic Envs preferentially replicated in peripheral blood mononuclear cells (PBMC) and a CD4 T-cell line compared to monocyte-derived macrophages, or dendritic cells (DC). In a model of trans infection from monocyte-derived dendritic cells to T cells, chimeric virus from acute Envs achieved significantly lower titers compared to chronic Envs. Challenge of primary human mucosal tissues revealed significantly higher levels of replication in chronic Env-expressing virus in rectal tissue compared to cervical and penile tissues and enhanced replication in tonsillar tissue relative to acute Envs. In agreement with data from the DC to T-cell trans infection assay, chronic Env-chimeric virus pools were transmitted more efficiently by migratory cells from cervical and penile tissues to CD4(+) T cells than individual acute Env chimeras. These data indicate that virus with HIV-1 Envs of transmitted acute infections preferentially replicate in T cells rather than macrophages or dendritic cells and are less efficiently transmitted from antigen-presenting cells to CD4 T cells than chronic Envs. Such properties together with chemokine (C-C motif) receptor 5 (CCR5) use may confer an advantage for transmission.

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

In the absence of a protective vaccine against human immunodeficiency virus (HIV), there is an urgent need for the development of effective topical microbicides to prevent HIV infection. Candidate vaginal microbicides should provide protection against circulating strains, be cheap, stable on storage, safe and easy to use. Here we describe a detailed study of the safety and efficacy of Cyanovirin-N (CV-N) in vitro, and in an ex vivo model of female genital tissue explants. CV-N demonstrated potent activity in the low nanomolar range against laboratory and primary isolates. Activity was related to the affinity of CV-N for binding to whole virions as determined by acoustic resonance. Potent activity was also observed against cell-associated HIV-1, although slightly reduced. CV-N activity in the presence of whole semen was reduced by 7-10-fold, although it remained in the low nanomolar range and was minimally modified by the presence of Candida albicans. Furthermore, CV-N potently inhibited infection of ectocervical explants and virus dissemination by tissue-emigrating cells. In peripheral blood mononuclear cell (PBMC) assays, CV-N was shown to have some mitogenic activity following 3 days exposure to compound, and this was associated with a modest increase in expression of gamma interferon, stromal cell-derived factor 1beta and interleukin 4. However, 2 h exposure to CV-N had no effect on cytokine expression in PBMC or tissue explant culture over a 24 h period, suggesting that the potential for inflammation is low. Data presented here indicate that targeting HIV envelope glycoproteins may provide an effective strategy to prevent HIV-1 infection mediated by either cell-free virus or infected cells.

Several studies have shown the importance of evaluating Recent Thymic Emigrants (RTEs) by quantification of T cell receptor-rearrangement excision circles (TRECs), as a measure of de novo T cell generation during human immunodeficiency virus-1 (HIV-1) infection. To determine whether acute viral infection may have an impact on TRECs, cynomolgus monkeys (Macaca fascicularis) were infected intrarectally with simian human immunodeficiency virus (SHIV) 89.6P(cy11) and the number of signal-joint (sj) TRECs was determined in purified CD4+ and CD8+ populations for up to 28 weeks post-infection. Four weeks after infection, TRECs levels significantly decreased in both CD3+ CD4+ and in CD3+ CD8+ T lymphocytes of infected monkeys, whereas they remained unchanged in uninfected animals. This reduction was followed by a progressive TRECs number recovery in CD3+ CD4+ T lymphocytes that positively correlated with changes in the levels of circulating CD3+ CD4+ T cells. In the CD3+ CD8+ T cell subset, TRECs number remained significantly low and inversely correlated with the increase in the percentages of CD3+ CD8+ T cells. These data suggest that SHIV89.6P(cy11) intrarectal infection of cynomolgus monkeys differently affects TRECs content in CD3+ CD4+ and CD3+ CD8+ T cell subsets.

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.

Genetic immunization using viral vectors provides an effective means to elicit antigen-specific cellular immune responses. Several viral vectors have proven efficacious in inducing immune responses after direct injection in vivo. Among them, recombinant, self-inactivating lentiviral vectors are very attractive delivery systems, as they are able to efficiently transduce into and express foreign genes in a wide variety of mammalian cells. A self-inactivating lentiviral vector was evaluated for the delivery of human immunodeficiency virus 1 (HIV-1) envelope sequences in mice in order to elicit specific immune responses. With this aim, BALB/c mice were immunized with a single injection of self-inactivating lentiviral vectors carrying either the full-length HIV-1(HXB2) Rev/Env (TY2-IIIBEnv) or the codon-optimized HIV-1(JR-FL) gp120 (TY2-JREnv) coding sequence. Both vectors were able to elicit specific cellular responses efficiently, as measured by gamma interferon ELISPOT and chromium-release assays, upon in vitro stimulation of splenocytes from BALB/c immunized mice. However, only the TY2-JREnv-immunized mice were able to elicit specific humoral responses, measured as anti-gp120 antibody production. These data provide the first evidence that a single, direct, in vivo administration of a lentiviral vector encoding a viral gene might represent a useful strategy for vaccine development.