Gp120 V3 Research Articles

A VRC13-like bNAb response is associated with complex escape pathways in HIV-1 envelope.

The rational design of HIV-1 immunogens to trigger the development of broadly neutralizing antibodies (bNAbs) requires understanding the viral evolutionary pathways influencing this process. An acute HIV-1-infected individual exhibiting >50% plasma neutralization breadth developed neutralizing antibody specificities against the CD4-binding site (CD4bs) and V1V2 regions of Env gp120. Comparison of pseudoviruses derived from early and late autologous env sequences demonstrated the development of >2 log resistance to VRC13 but not to other CD4bs-specific bNAbs. Mapping studies indicated that the V3 and CD4-binding loops of Env gp120 contributed significantly to developing resistance to the autologous neutralizing response and that the CD4-binding loop (CD4BL) specifically was responsible for the developing resistance to VRC13. Tracking viral evolution during the development of this cross-neutralizing CD4bs response identified amino acid substitutions arising at only 4 of 11 known VRC13 contact sites (K282, T283, K421, and V471). However, each of these mutations was external to the V3 and CD4BL regions conferring resistance to VRC13 and was transient in nature. Rather, complete resistance to VRC13 was achieved through the cooperative expression of a cluster of single amino acid changes within and immediately adjacent to the CD4BL, including a T359I substitution, exchange of a potential N-linked glycosylation (PNLG) site to residue S362 from N363, and a P369L substitution. Collectively, our data characterize complex HIV-1 env evolution in an individual developing resistance to a VRC13-like neutralizing antibody response and identify novel VRC13-associated escape mutations that may be important to inducing VRC13-like bNAbs for lineage-based immunogens.IMPORTANCEThe pursuit of eliciting broadly neutralizing antibodies (bNAbs) through vaccination and their use as therapeutics remains a significant focus in the effort to eradicate HIV-1. Key to our understanding of this approach is a more extensive understanding of bNAb contact sites and susceptible escape mutations in HIV-1 envelope (env). We identified a broad neutralizer exhibiting VRC13-like responses, a non-germline restricted class of CD4-binding site antibody distinct from the well-studied VRC01-class. Through longitudinal envelope sequencing and Env-pseudotyped neutralization assays, we characterized a complex escape pathway requiring the cooperative evolution of four amino acid changes to confer complete resistance to VRC13. This suggests that VRC13-class bNAbs may be refractory to rapid escape and attractive for therapeutic applications. Furthermore, the identification of longitudinal viral changes concomitant with the development of neutralization breadth may help identify the viral intermediates needed for the maturation of VRC13-like responses and the design of lineage-based immunogens.

Intermediate conformations of CD4-bound HIV-1 Env heterotrimers

HIV-1 envelope (Env) exhibits distinct conformational changes in response to host receptor (CD4) engagement. Env, a trimer of gp120 and gp41 heterodimers, has been structurally characterized in a closed, prefusion conformation with closely associated gp120s and coreceptor binding sites on gp120 V3 hidden by V1V2 loops1–4 and in fully saturated CD4-bound open Env conformations with changes including outwardly rotated gp120s and displaced V1V2 loops3–9. To investigate changes resulting from substoichiometric CD4 binding, we solved single-particle cryo-electron microscopy (cryo-EM) structures of soluble, native-like heterotrimeric Envs bound to one or two CD4 molecules. Most of the Env trimers bound to one CD4 adopted the closed, prefusion Env state, with a minority exhibiting a heterogeneous partially open Env conformation. When bound to two CD4s, the CD4-bound gp120s exhibited an open Env conformation including a four-stranded gp120 bridging sheet and displaced gp120 V1V2 loops that expose the coreceptor sites on V3. The third gp120 adopted an intermediate, occluded-open state10 that showed gp120 outward rotation but maintained the prefusion three-stranded gp120 bridging sheet with only partial V1V2 displacement and V3 exposure. We conclude that most of the engagements with one CD4 molecule were insufficient to stimulate CD4-induced conformational changes, whereas binding two CD4 molecules led to Env opening in CD4-bound protomers only. The substoichiometric CD4-bound soluble Env heterotrimer structures resembled counterparts derived from a cryo-electron tomography study of complexes between virion-bound Envs and membrane-anchored CD4 (ref. 11), validating their physiological relevance. Together, these results illuminate intermediate conformations of HIV-1 Env and illustrate its structural plasticity.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

CXCR4 Recognition by L- and D-Peptides Containing the Full-Length V3 Loop of HIV-1 gp120.

Human immunodeficiency virus-1 (HIV-1) recognizes one of its principal coreceptors, CXC chemokine receptor 4 (CXCR4), on the host cell via the third variable loop (V3 loop) of HIV-1 envelope glycoprotein gp120 during the viral entry process. Here, the mechanism of the molecular recognition of HIV-1 gp120 V3 loop by coreceptor CXCR4 was probed by synthetic peptides containing the full-length V3 loop. The two ends of the V3 loop were covalently linked by a disulfide bond to form a cyclic peptide with better conformational integrity. In addition, to probe the effect of the changed side-chain conformations of the peptide on CXCR4 recognition, an all-D-amino acid analog of the L-V3 loop peptide was generated. Both of these cyclic L- and D-V3 loop peptides displayed comparable binding recognition to the CXCR4 receptor, but not to another chemokine receptor, CCR5, suggesting their selective interactions with CXCR4. Molecular modeling studies revealed the important roles played by many negative-charged Asp and Glu residues on CXCR4 that probably engaged in favorable electrostatic interactions with the positive-charged Arg residues present in these peptides. These results support the notion that the HIV-1 gp120 V3 loop-CXCR4 interface is flexible for ligands of different chiralities, which might be relevant in terms of the ability of the virus to retain coreceptor recognition despite the mutations at the V3 loop.

Prediction of Coreceptor Tropism in HIV-1 Subtype C in Botswana.

It remains unknown whether the C-C motif chemokine receptor type 5 (CCR5) coreceptor is still the predominant coreceptor used by Human Immunodeficiency Virus-1 (HIV-1) in Botswana, where the HIV-1 subtype C predominates. We sought to determine HIV-1C tropism in Botswana using genotypic tools, taking into account the effect of antiretroviral treatment (ART) and virologic suppression. HIV-1 gp120 V3 loop sequences from 5602 participants were analyzed for viral tropism using three coreceptor use predicting algorithms/tools: Geno2pheno, HIV-1C Web Position-Specific Score Matrices (WebPSSM) and the 11/25 charge rule. We then compared the demographic and clinical characteristics of people living with HIV (PLWH) harboring R5- versus X4-tropic viruses using χ2 and Wilcoxon rank sum tests for categorical and continuous data analysis, respectively. The three tools congruently predicted 64% of viruses as either R5-tropic or X4-tropic. Geno2pheno and the 11/25 charge rule had the highest concordance at 89%. We observed a significant difference in ART status between participants harboring X4- versus R5-tropic viruses. X4-tropic viruses were more frequent among PLWH receiving ART (χ2 test, p = 0.03). CCR5 is the predominant coreceptor used by HIV-1C strains circulating in Botswana, underlining the strong potential for CCR5 inhibitor use, even in PLWH with drug resistance. We suggest that the tools for coreceptor prediction should be used in combination.

Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.

Imprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified. In this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes. In the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro. Our data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.

HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop

The human immunodeficiency virus type 1 (HIV-1) recognizes one of its principal coreceptors, the CXC chemokine receptor 4 (CXCR4) on the host cell via the third variable loop (V3 loop) of HIV-1 envelope glycoprotein gp120 during the viral entry process. Here, we investigated the stereochemical mechanism of the molecular recognition of HIV-1 gp120 V3 loop with coreceptor CXCR4 by using peptide probes containing important fragments of the V3 loop. The tip and base/stem fragments of the V3 loop critical for V3 loop function were linked individually with the fragment derived from another CXCR4's chemokine ligand, vMIP-II to generate nanomolar affinity peptide probes of the interactions of CXCR4-V3 loop fragments. When the amino acid residues of the V3 loop fragments in these combinational peptides were changed from L-to D-configurations, the resulting peptides remarkably retained or had even enhanced recognition by CXCR4 as shown by competitive ligand-receptor binding. The ability of these peptides, regardless of the different l- or d-amino acids used, in binding CXCR4 and antagonizing CXCR4 functions was demonstrated by their blockade of calcium influx, cell migration, and CXCR4 internalization triggered by the activation of CXCR4 signaling by its endogenous ligand SDF-1α. The structural mechanisms of CXCR4 interactions with these peptides were examined with site-directed mutagenesis and molecular modeling. These results indicate that CXCR4's interface with key segments of HIV-1 gp120 V3 loop is flexible in terms of stereospecificity of ligand-receptor interaction which may have implication on understanding the viral entry mechanism and how the virus evades immune detection with V3 loop mutations and retains effective recognition of the host cell's coreceptor.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

Design, synthesis, and evaluation of HIV-1 entry inhibitors based on broadly neutralizing antibody 447-52D and gp120 V3loop interactions

The third variable loop region (V3 loop) on gp120 plays an important role in cellular entry of HIV-1. Its interaction with the cellular CD4 and coreceptors is an important hallmark in facilitating the bridging by gp41 and subsequent fusion of membranes for transfer of viral genetic material. Further, the virus phenotype determines the cell tropism via respective co– receptor binding. Thus, coreceptor binding motif of envelope is considered to be a potent anti-viral drug target for viral entry inhibition. However, its high variability in sequence is the major hurdle for developing inhibitors targeting the region. In this study, we have used an in silico Virtual Screening and “Fragment-based” method to design small molecules based on the gp120 V3 loop interactions with a potent broadly neutralizing human monoclonal antibody, 447-52D. From the in silico analysis a potent scaffold, 1,3,5-triazine was identified for further development. Derivatives of 1,3,5-triazine with specific functional groups were designed and synthesized keeping the interaction with co-receptor intact. Finally, preliminary evaluation of molecules for HIV-1 inhibition on two different virus strains (clade C, clade B) yielded IC50 < 5.0 μM. The approach used to design molecules based on broadly neutralizing antibody, was useful for development of target specific potent antiviral agents to prevent HIV entry. The study reported promising inhibitors that could be further developed and studied.

Double Arylation of the Indole Side Chain of Tri- and Tetrapodal Tryptophan Derivatives Renders Highly Potent HIV-1 and EV-A71 Entry Inhibitors†.

We have recently described a new generation of potent human immunodeficiency virus (HIV) and EV-A71 entry inhibitors. The prototypes contain three or four tryptophan (Trp) residues bearing an isophthalic acid moiety at the C2 position of each side-chain indole ring. This work is now extended by both shifting the position of the isophthalic acid to C7 and synthesizing doubly arylated C2/C7 derivatives. The most potent derivative (50% effective concentration (EC50) HIV-1, 6 nM; EC50 EV-A71, 40 nM), 33 (AL-518), is a C2/C7 doubly arylated tetrapodal compound. Its superior anti-HIV potency with respect to the previous C2-arylated prototype is in consonance with its higher affinity for the viral gp120. 33 (AL-518) showed comparable antiviral activities against X4 and R5 HIV-1 strains and seems to interact with the tip and base of the gp120 V3 loop. Taken together, these findings support the interest in 33 (AL-518) as a useful new prototype for anti-HIV/EV71 drug development.

Exploring Viral Interference Using Peptides: Molecular Determinants of HIV-1 Inhibition by a Peptide Derived from Human Pegivirus-1 Envelope Protein E2.

Co‐infection with the human pegivirus 1 (HPgV‐1) often has a beneficial effect on disease progression in HIV‐1‐infected individuals. Several HPgV‐1 proteins and peptides, including a 20‐mer peptide (P6‐2) derived from the N‐terminal region of the HPgV‐1 surface protein E2, have been associated with this phenomenon, which is referred to as viral interference. We identified the cysteine residues, the hydrophobic core tetrapeptide, as well as the C‐terminal negative charge as key factors for the HIV‐1 inhibitory activity of P6‐2. Analysis of mutations in P6‐2‐resistant HIV‐1 indicated a binding site for the peptide in the HIV‐1 envelope glycoprotein gp120. In fact, P6‐2 was shown to bind to soluble gp120, as well as to a peptide presenting the gp120 V3 loop. Furthermore, the HIV‐1 inhibitory activity of P6‐2 could be revoked by the V3 loop peptide, thus indicating a molecular mechanism that involves interaction of P6‐2 with the gp120 V3 loop.

The V2 loop of HIV gp120 delivers costimulatory signals to CD4+ T cells through Integrin α4β7 and promotes cellular activation and infection

Acute HIV infection is characterized by rapid viral seeding of immunologic inductive sites in the gut followed by the severe depletion of gut CD4+ T cells. Trafficking of α4β7-expressing lymphocytes to the gut is mediated by MAdCAM, the natural ligand of α4β7 that is expressed on gut endothelial cells. MAdCAM signaling through α4β7 costimulates CD4+ T cells and promotes HIV replication. Similar to MAdCAM, the V2 domain of the gp120 HIV envelope protein binds to α4β7 In this study, we report that gp120 V2 shares with MAdCAM the capacity to signal through α4β7 resulting in CD4+ T cell activation and proliferation. As with MAdCAM-mediated costimulation, cellular activation induced by gp120 V2 is inhibited by anti-α4β7 monoclonal antibodies (mAbs). It is also inhibited by anti-V2 domain antibodies including nonneutralizing mAbs that recognize an epitope in V2 that has been linked to reduced risk of acquisition in the RV144 vaccine trial. The capacity of the V2 domain of gp120 to mediate signaling through α4β7 likely impacts early events in HIV infection. The capacity of nonneutralizing V2 antibodies to block this activity reveals a previously unrecognized mechanism whereby such antibodies might impact HIV transmission and pathogenesis.

HIV-1 acquisition in a man with ulcerative colitis on anti-α4β7 mAb vedolizumab treatment.

HIV-1 acquisition in a man with ulcerative colitis on anti-α4β7 mAb vedolizumab treatment.

Identification of novel molecular determinants of co-receptor usage in HIV-1 subtype F V3 envelope sequences

HIV-1 determinants of coreceptor usage within the gp120 V3 loop have been broadly studied over the past years. This information has led to the development of state-of the-art bioinformatic tools that are useful to predict co-receptor usage based on the V3 loop sequence mainly of subtypes B, C and A. However, these methods show a poor performance for subtype F V3 loops, which are found in an increasing number of HIV-1 strains worldwide. In the present work we investigated determinants of viral tropisms in the understudied subtype F by looking at genotypic and structural information of coreceptor:V3 loop interactions in a novel group of 40 subtype F V3 loops obtained from HIV-1 strains phenotypically characterized either as syncytium inducing or non-syncytium inducing by the MT-2 assay. We provide novel information about estimated interactions energies between a set of V3 loops with known tropism in subtype F, that allowed us to improve predictions of the coreceptor usage for this subtype. Understanding genetic and structural features underlying HIV coreceptor usage across different subtypes is relevant for the rational design of preventive and therapeutic strategies aimed at limiting the HIV-1 epidemic worldwide.

Influence of the HIV GWG variant in the HIV infection progression in mono and HCV coinfected patients.

The HIV subtype B is the most frequent in Brazil. The HIV subtype B’ codes the amino acids glicine-tryptophan-glicine (GWG) instead of glicine-proline-glicine on the tip of gp120 V3 loop. This variant was associated to a slower HIV progression in mono-infected patients; however, there is no information in coinfected patients. This study evaluated the infection progression of HIV variant B’ on the hepatitis C virus presence. RNA isolated from plasma of the 601 infected patients were used to human immunodeficiency virus (HIV) subtyping and to classify the virus according their syncytium-inducing ability. The HIV infection progression was evaluated by clinical and laboratorial data. The results showed a significant association between HIV B’ variant and CD4 count and time of AIDS in HIV mono-infected patients. Notwithstanding the fact that we did not find a direct association between GWG variant and AIDS and in HIV coinfected patients no mitigating effect due to GWG presence was found. We did observe that the association between GWG variant and CD4 counts is lost in coinfected patients. This is first work showing influence of the HIV GWG variant in coinfected patients. Nevertheless, the presence of the GWG variant can indicate a better prognostic in the mono-infected patients.

Super Potent Bispecific Llama VHH Antibodies Neutralize HIV via a Combination of gp41 and gp120 Epitopes.

Broad and potent neutralizing llama single domain antibodies (VHH) against HIV-1 targeting the CD4 binding site (CD4bs) have previously been isolated upon llama immunization. Here we describe the epitopes of three additional VHH groups selected from phage libraries. The 2E7 group binds to a new linear epitope in the first heptad repeat of gp41 that is only exposed in the fusion-intermediate conformation. The 1B5 group competes with co-receptor binding and the 1F10 group interacts with the crown of the gp120 V3 loop, occluded in native Env. We present biophysical and structural details on the 2E7 interaction with gp41. In order to further increase breadth and potency, we constructed bi-specific VHH. The combination of CD4bs VHH (J3/3E3) with 2E7 group VHH enhanced strain-specific neutralization with potencies up to 1400-fold higher than the mixture of the individual VHHs. Thus, these new bivalent VHH are potent new tools to develop therapeutic approaches or microbicide intervention.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

The Dual Role of HIV-1 gp120 V3 Loop-Induced Autophagy in the Survival and Apoptosis of the Primary Rat Hippocampal Neurons.

HIV-1 gp120, an important subunit of the envelope spikes that decorate the surface of virions, is known to play a vital role in neuronal injury during HIV-1-associated neurocognitive disorder (HAND), although the pathological mechanism is not fully understood. Our previous studies have suggested that the V3 loop of HIV-1 gp120 (HIV-1 gp120 V3 loop) can induce neuronal apoptosis in the hippocampus, resulting in impairment in spatial learning and memory in Sprague-Dawley (SD) rats. In this study, we demonstrated that autophagy was significantly increased in rat primary hippocampal neurons in response to treatment of HIV-1 gp120 V3 loop. Importantly, HIV-1 gp120 V3 loop-induced autophagy played a dual role in the cell survival and death. An increase in autophagy for a short period inhibited apoptosis of neurons, while persistent autophagy over an extended period of time played a detrimental role by augmenting the apoptotic cascade in rat primary hippocampal neurons. In addition, we found that the HIV-1 gp120 V3 loop induced autophagy via AMPK/mTOR-dependent and calpain/mTOR-independent pathways, and the ERK/mTOR pathway plays a partial role. These findings provide evidence that HIV-1-induced autophagy plays a dual role in the survival and apoptosis of the primary rat hippocampal neurons and persistent autophagy may contribute to the pathogenesis of HAND, and autophagy modulation may represent a potential therapeutic strategy for reducing neuronal damage in HAND.

PO 8411 MOLECULAR CHARACTERISATION OF GP41 AND GP120 V3 LOOP IN HIV-1C PATIENTS FAILING SALVAGE THERAPY IN BOTSWANA

BackgroundTriple class drug-resistant HIV-1 infection remains a global challenge in individuals with extensive antiretroviral treatment (ART) experience, in terms of high mortality and probability of onward transmission. New therapeutic options within old and new drug classes are therefore essential. We determined if patients failing salvage therapy in Botswana are eligible for maraviroc (MVC) and enfuvirtide (T20) viral entry inhibitors based on the coreceptor usage and drug-resistant mutations in envelope gp120 and gp41.MethodsA total of 38 deep salvage patients were included in the analysis. We amplified and sequenced gp41 and V3 regions of HIV-1 envelope. Drug resistance mutations were analysed according to the IAS-USA 2017 reference mutation lists. Coreceptor usage was determined using PSSM and Geno2Pheno using a false-positive rate (FPR) of 10%.ResultsAmong 38 participants, 34 (89%) were successfully sequenced and amplified gp41 and 26 (68%) gp120 V3 loop sequences were obtained. Major T20 mutation G36S was obtained in 1/34 samples (5.8%) within the study population. Polymorphisms I169V(97%), I135L(100%), E151A(70.6%) and N42S(70.6%) were detected in HR1 and HR2 of gp41. CXCR4 coreceptor associated use, mutation L34M in gp41 HR1 was detected in 2 samples (5%). Analysis of coreceptor usage showed (17/26) 65.4% use of CCR5, and a (9/26) 34.6% use of the CXCR4 coreceptor.ConclusionA moderately high proportion of treatment-experienced (deep salvage) participants had CXCR4 coreceptor using strains. The use of maraviroc in Botswana would require coreceptor tropism testing. Non-T20 treatment experience in Botswana reduces the prevalence of the major mutations that confer resistance to the drug. T20 is therefore a potential alternative drug for patients failing salvage therapy in Botswana.

HIV-1 vaccination by needle-free oral injection induces strong mucosal immunity and protects against SHIV challenge

The oral mucosa is an attractive site for mucosal vaccination, however the thick squamous epithelium limits antigen uptake. Here we utilize a modified needle-free injector to deliver immunizations to the sublingual and buccal (SL/B) tissue of rhesus macaques. Needle-free SL/B vaccination with modified vaccinia Ankara (MVA) and a recombinant trimeric gp120 protein generates strong vaccine-specific IgG responses in serum as well as vaginal, rectal and salivary secretions. Vaccine-induced IgG responses show a remarkable breadth against gp70-V1V2 sequences from multiple clades of HIV-1. In contrast, topical SL/B immunizations generates minimal IgG responses. Following six intrarectal pathogenic SHIV-SF162P3 challenges, needle-free but not topical immunization results in a significant delay of acquisition of infection. Delay of infection correlates with non-neutralizing antibody effector function, Env-specific CD4+ T-cell responses, and gp120 V2 loop specific antibodies. These results demonstrate needle-free MVA/gp120 oral vaccination as a practical and effective route to induce protective immunity against HIV-1.