Developing an HIV-1 vaccine that can elicit antibodies to prevent infection has been a formidable challenge. Although no single immunogen has generated antibodies that can neutralize diverse ...isolates, progress has been made in understanding (a) the structure of the HIV-1 envelope glycoprotein, which is targeted by neutralizing antibodies, (b) how HIV-1 evades antibodies made by an infected host, and (c) how rare monoclonal antibodies can exhibit broadly neutralizing activity. Advances in structural and molecular biology coupled with new approaches to isolate neutralizing antibodies from HIV-1-infected individuals are enhancing our understanding of what humoral immune responses will be required for a vaccine. This review summarizes progress in understanding the host antibody response to HIV-1 and current strategies for applying this information to develop an effective vaccine.
The initial step in target cell infection by human, and the closely related simian immunodeficiency viruses (HIV and SIV, respectively) occurs with the binding of trimeric envelope glycoproteins ...(Env), composed of heterodimers of the viral transmembrane glycoprotein (gp41) and surface glycoprotein (gp120) to target T-cells. Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogen-based vaccines to combat HIV/AIDS. Previous analyses of intact HIV-1 BaL virions have already resulted in structures of trimeric Env in unliganded and CD4-liganded states at ~20 Å resolution. Here, we show that the molecular architectures of trimeric Env from SIVmneE11S, SIVmac239 and HIV-1 R3A strains are closely comparable to that previously determined for HIV-1 BaL, with the V1 and V2 variable loops located at the apex of the spike, close to the contact zone between virus and cell. The location of the V1/V2 loops in trimeric Env was definitively confirmed by structural analysis of HIV-1 R3A virions engineered to express Env with deletion of these loops. Strikingly, in SIV CP-MAC, a CD4-independent strain, trimeric Env is in a constitutively "open" conformation with gp120 trimers splayed out in a conformation similar to that seen for HIV-1 BaL Env when it is complexed with sCD4 and the CD4i antibody 17b. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses.
The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body ...simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, (64)Cu-labeled SIV Gp120-specific antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.
The H.sub.196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM.sub.195 ), ...and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q.sub.196 . In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q.sub.196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q.sub.196 residue in Nef uniformly also encode an upstream R.sub.191 residue. We show that R.sub.191 restores the ability of Nef to downregulate tetherin in the presence of Q.sub.196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q.sub.196 commonly evolves to H.sub.196 in vivo, suggesting a fitness cost. R.sub.191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q.sub.196.
HIV-1 infection depends on effective viral entry mediated by the interaction of its envelope (Env) glycoprotein with specific cell surface receptors. Protective antiviral antibodies generated by ...passive or active immunization must prevent these interactions. Because the HIV-1 Env is highly variable, attention has also focused on blocking the HIV-1 primary cell receptor CD4. We therefore analyzed the in vivo protective efficacy of three potent neutralizing monoclonal antibodies (mAbs) to HIV-1 Env compared to an antibody against the CD4 receptor. Protection was assessed after mucosal challenge of rhesus macaques with simian/HIV (SHIV). Despite its comparable or greater neutralization potency in vitro, the anti-CD4 antibody did not provide effective protection in vivo, whereas the HIV-1-specific mAbs VRC01, 10E8, and PG9, targeting the CD4 binding site, membrane-proximal, and V1V2 glycan Env regions, respectively, conferred complete protection, albeit at different relative potencies. These findings demonstrate the protective efficacy of broadly neutralizing antibodies directed to the HIV-1 Env and suggest that targeting the HIV-1 Env is preferable to the cell surface receptor CD4 for the prevention of HIV-1 transmission.
An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ...ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4-1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4
T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4-1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4-1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4
T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.
HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these ...peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans.
Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic stem-cell transplantation (HSCT). The chemokine receptor CCR5 appears to play a role in alloreactivity. We ...tested whether CCR5 blockade would be safe and limit GVHD in humans.
We tested the in vitro effect of the CCR5 antagonist maraviroc on lymphocyte function and chemotaxis. We then enrolled 38 high-risk patients in a single-group phase 1 and 2 study of reduced-intensity allogeneic HSCT that combined maraviroc with standard GVHD prophylaxis.
Maraviroc inhibited CCR5 internalization and lymphocyte chemotaxis in vitro without impairing T-cell function or formation of hematopoietic-cell colonies. In 35 patients who could be evaluated, the cumulative incidence rate (±SE) of grade II to IV acute GVHD was low at 14.7±6.2% on day 100 and 23.6±7.4% on day 180. Acute liver and gut GVHD were not observed before day 100 and remained uncommon before day 180, resulting in a low cumulative incidence of grade III or IV GVHD on day 180 (5.9±4.1%). The 1-year rate of death that was not preceded by disease relapse was 11.7±5.6% without excessive rates of relapse or infection. Serum from patients receiving maraviroc prevented CCR5 internalization by CCL5 and blocked T-cell chemotaxis in vitro, providing evidence of antichemotactic activity.
In this study, inhibition of lymphocyte trafficking was a specific and potentially effective new strategy to prevent visceral acute GVHD. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00948753.).
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