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Flagellin’s potential as a vaccine adjuvant has been increasingly explored over the last three decades. Monomeric flagellin proteins are the only known agonists of Toll-like receptor 5 (TLR5). This interaction evokes a pro-inflammatory state that impacts upon both innate and adaptive immunity. While pathogen associated molecular patterns (PAMPs) like flagellin have been used as stand-alone adjuvants that are co-delivered with antigen, some investigators have demonstrated a distinct advantage to incorporating antigen epitopes within the structure of flagellin itself. This approach has been particularly effective in enhancing humoral immune responses. We sought to use flagellin as both scaffold and adjuvant for HIV gp41 with the aim of eliciting antibodies to the membrane proximal external region (MPER). Accordingly, we devised a straightforward step-wise approach to select flagellin-antigen fusion proteins for gene-based vaccine development. Using plasmid DNA vector-based expression in mammalian cells, we demonstrate robust expression of codon-optimized full length and hypervariable region-deleted constructs of Salmonella enterica subsp. enterica serovar Typhi flagellin (FliC). An HIV gp41 derived sequence including the MPER (gp41607–683) was incorporated into various positions of these constructs and the expressed fusion proteins were screened for effective secretion, TLR5 agonist activity and adequate MPER antigenicity. We show that incorporation of gp41607–683 into a FliC-based scaffold significantly augments gp41607–683 immunogenicity in a TLR5 dependent manner and elicits modest MPER-specific humoral responses in a mouse model.

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER’s hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.

Continued high rates of HIV-1 transmission have mandated vigorous interest in a variety of approaches to prevent new infections including the testing of topical microbicides and prophylactic vaccines. Though the ideal prophylactic agent would result in sterile immunity, it has been theorized that even in the face of infection, a reduction in levels of plasma viremia postinfection may result in reduced transmissions at both the individual and population level.1,2 Recent discouraging results in trials of microbicides and vaccines have further fueled interest in the use of antiretrovirals (ARVs) to prevent infection. ARV therapy has not only increased survival for HIV-infected patients3-5 but has also been used to prevent both vertical transmission6 and transmission due to occupational exposure.7 The demonstrated efficacy of tenofovir DF (TDF) in the treatment of HIV-1 infection,8 the lack of reported serious adverse events associated with its use,8 its long half-life,9,10 and the fact that HIV-1 strains resistant to TDF tend to be less replication competent than wild-type viruses11 make this ARV agent an ideal candidate for pre- and postexposure prophylaxis (PrEP and PEP). Animal studies have provided evidence that PrEP and PEP with TDF alone, or in combination with emtricitabine (FTC), protect against simian immunodeficiency virus (SIV) and Simian HIV (SHIV) transmission.12-17 Furthermore, animals that become infected despite receiving TDF as PrEP or PEP showed delayed onset of viremia and seroconversion,14,17,18 demonstrating that even in the case of incomplete protection against infection, PrEP/PEP treatment may lead to attenuated acute infection. As transmission risk is a function of plasma viremia and acute infection represents a period during which transmission risk is high,19 such an effect may further act to prevent forward transmissions. Here, we report a case of an individual infected with a drug-susceptible virus despite the intermittent use of fixed-dose combination TDF/FTC (Truvada; Gilead Sciences, Foster City, CA) as PrEP/PEP. As seen in animal models, the resultant infection displayed an attenuated course that could not be attributed to known modifying host factors, suggesting that despite inability to completely prevent infection, antiviral therapy may indeed modulate acute and early HIV-1 infection with resultant beneficial effects on preventing forward infections.

During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+T cells during AEI. HIV-1 RNA was detected in both “activated” and“ nonactivated” mucosal CD4+T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+T cells. Subsequently, ongoing infection of susceptible CD4+T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion.

Transmitted resistance to antiretroviral drugs in acute and early HIV-1 infection has been well documented, although overall trends vary depending on geography and cohort characteristics. To describe the changing pattern of transmitted drug-resistant HIV-1 in a well-defined cohort in New York City, a total of 361 patients with acute or recent HIV-1 infection were prospectively studied over a decade (1995-2004) with respect to HIV-1 genotypes and longitudinal T-cell subsets and HIV-1 RNA levels. The prevalence of overall transmitted resistance changed from 13.2% to 24.1% (P = 0.11) during the periods 1995 to 1998 and 2003 to 2004. Nonnucleoside reverse transcriptase inhibitor resistance prevalence increased significantly from 2.6% to 13.4% (P = 0.007) during the same periods, whereas prevalence of multidrug-resistant virus shifted from 2.6% to 9.8% (P = 0.07) but did not achieve statistical significance. A comparable immunologic and virologic response of appropriately treated individuals was observed regardless of viral drug susceptibility status, suggesting that initial combination therapy guided by baseline resistance testing in the case of acute and early infection may result in a favorable treatment response even in the case of a drug-resistant virus. These data have important implications for selection of empiric first-line regimens for treatment of acutely infected antiretroviral-naive individuals and reinforce the need for baseline resistance testing in acute and early HIV-1 infection.

Summary Background Rapid progression to AIDS after acute HIV-1 infection, though uncommon, has been noted, as has the transmission of multidrug resistant viruses. Here, we describe a patient in whom these two factors arose concomitantly and assess the effects. Methods We did a case study of a patient with HIV-1 seroconversion. We genotyped the virus and host genetic markers by PCR and nucleotide sequencing. To ascertain the drug susceptibility of our patient's HIV-1 we did phenotypic studies with the PhenoSense assay. We assessed viral coreceptor use via syncytium formation in vitro and with a modified PhenoSense assay. Findings Our patient seems to have been recently infected by a viral variant of HIV-1 resistant to multiple classes of antiretroviral drugs. Furthermore, his virus population is dual tropic for cells that express CCR5 or CXCR4 coreceptor. The infection has resulted in progression to symptomatic AIDS in 4–20 months. Interpretation The intersection of multidrug resistance and rapid development of AIDS in this patient is of concern, especially in view of his case history, which includes high-risk sexual contacts and use of metamfetamine. The public health ramifications of such a case are great.