HIV-related drivers of sexual compulsivity and sexuality in sexual minority men who use methamphetamine.

Although co-occurring methamphetamine (meth) use and HIV amplify the risk for neuropsychiatric comorbidities, the underlying neuroimmune mechanisms are not well characterized. We examined whether a detectable viral load and dysregulated metabolism of amino acid precursors for neurotransmitters predicted subsequent levels of sexual compulsivity and sexual sensation seeking. This 15-month longitudinal study enrolled 110 sexual minority men (SMM) living with HIV who had biologically confirmed meth use (i.e., reactive urine or hair toxicology results). Peripheral venous blood samples collected at baseline, 6 months, 12 months, and 15 months were used to measure a detectable viral load (> 40 copies/mL), the kynurenine/tryptophan (K/T) ratio, and the phenylalanine/tyrosine (P/T) ratio. The K/T and P/T ratios index dysregulated serotonin and catecholamine (e.g., dopamine) synthesis, respectively. In a cross-lagged panel model, a detectable viral load at 6 months predicted greater sexual compulsivity at 12 months after adjusting for prior levels of sexual compulsivity and recent stimulant use (ß = 0.26, p = 0.046). A greater P/T ratio at baseline predicted decreased sexual sensation seeking at 6 months (ß = - 0.25, p = 0.004) after adjusting for baseline sexual sensation seeking and recent stimulant use. Taken together, HIV replication and dysregulated catecholamine synthesis could potentiate sexual compulsivity while decreasing sexual pleasure in SMM who use meth.

AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.

Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 µg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 µg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 µg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.

Anti-drug Antibody Responses Impair Prophylaxis Mediated by AAV-Delivered HIV-1 Broadly Neutralizing Antibodies.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs.

CD4-Induced Antibodies Promote Association of the HIV-1 Envelope Glycoprotein with CD4-Binding Site Antibodies.

UNLABELLED: The HIV-1 envelope glycoprotein (Env) is a trimer of gp120/gp41 heterodimers that mediates viral entry. Env binds cellular CD4, an association which stabilizes a conformation favorable to its subsequent association with a coreceptor, typically CCR5 or CXCR4. The CD4- and coreceptor-binding sites serve as epitopes for two classes of HIV-1-neutralizing antibodies: CD4-binding site (CD4bs) and CD4-induced (CD4i) antibodies, respectively. Here we observed that, at a fixed total concentration, mixtures of the CD4i antibodies (E51 or 412d) and the CD4bs antibody VRC01 neutralized the HIV-1 isolates 89.6, ADA, SG3, and SA32 more efficiently than either antibody alone. We found that E51, and to a lesser extent 412d and 17b, promoted association of four CD4bs antibodies to the Env trimer but not to monomeric gp120. We further demonstrated that the binding of the sulfotyrosine-binding pocket by CCR5mim2-Ig was sufficient for promoting CD4bs antibody binding to Env. Interestingly, the relationship is not reciprocal: CD4bs antibodies were not as efficient as CD4-Ig at promoting E51 or 412d binding to Env trimer. Consistent with these observations, CD4-Ig, but none of the CD4bs antibodies tested, substantially increased HIV-1 infection of a CD4-negative, CCR5-positive cell line. We conclude that the ability of CD4i antibodies to promote VRC01 association with Env trimers accounts for the increase potency of VRC01 and CD4i antibody mixtures. Our data further suggest that potent CD4bs antibodies avoid inducing Env conformations that bind CD4i antibodies or CCR5. IMPORTANCE: Potent HIV-1-neutralizing antibodies can prevent viral transmission and suppress an ongoing infection. Here we show that CD4-induced (CD4i) antibodies, which recognize the conserved coreceptor-binding site of the HIV-1 envelope glycoprotein (Env), can increase the association of Env with potent broadly neutralizing antibodies that recognize the CD4-binding site (CD4bs antibodies). We further show that, unlike soluble forms of CD4, CD4bs antibodies poorly induce envelope glycoprotein conformations that efficiently bind CCR5. This study provides insight into the properties of potent CD4bs antibodies and suggests that, under some conditions, CD4i antibodies can improve their potency. These observations may be helpful to the development of vaccines designed to elicit specific antibody classes.

Interferon-induced transmembrane protein 3 is a type II transmembrane protein.

The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.

AAV-delivered eCD4-Ig protects rhesus macaques from high-dose SIVmac239 challenges.

A number of simian and simian human immunodeficiency viruses (SIV and SHIV, respectively) have been used to assess the efficacy of HIV-1 vaccine strategies. Among these, SIVmac239 is considered among the most stringent because, unlike SHIV models, its full genome has coevolved in its macaque host and its tier 3 envelope glycoprotein (Env) is exceptionally hard to neutralize. Here, we investigated the ability of eCD4-Ig, an antibody-like entry inhibitor that emulates the HIV-1 and SIV receptor and coreceptor, to prevent SIVmac239 infection. We show that rh-eCD4-IgI39N expressed by recombinant adeno-associated virus (AAV) vectors afforded four rhesus macaques complete protection from high-dose SIVmac239 challenges that infected all eight control macaques. However, rh-eCD4-IgI39N-expressing macaques eventually succumbed to serial escalating challenge doses that were 2, 8, 16, and 32 times the challenge doses that infected the control animals. Despite receiving greater challenge doses, these macaques had significantly lower peak and postpeak viral loads than the control group. Virus isolated from three of four macaques showed evidence of strong immune pressure from rh-eCD4-IgI39N, with mutations located in the CD4-binding site, which, in one case, exploited a point-mutation difference between rh-eCD4-IgI39N and rhesus CD4. Other escape pathways associated with clear fitness costs to the virus. Our data report effective protection of rhesus macaques from SIVmac239.