A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice.

BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.

Association of Alcohol Consumption With CD4 Recovery After Antiretroviral Therapy Initiation in St. Petersburg, Russia.

BACKGROUND: Delayed CD4 recovery after initiating antiretroviral therapy (ART) is a novel potential mechanism by which alcohol consumption leads to increased morbidity and mortality in people with HIV. We hypothesized that alcohol consumption at ART initiation is associated with slower CD4 recovery. METHODS: We retrospectively analyzed 2 pooled longitudinal alcohol/HIV cohorts (2014-2019) in St. Petersburg, Russia. Eligible participants initiated the first ART during parent studies; had alcohol consumption assessed by the blood biomarker, phosphatidylethanol (PEth), at the last research visit before ART initiation; and had ≥1 CD4 count measurement before and after initiating ART. Participants were stratified by low, moderate, and high PEth (<8, 8-80, and >80 ng/mL, respectively). We used random-effects piecewise linear regression models to estimate CD4 recovery, defined as CD4 count change per 30 days after ART initiation, by the alcohol group. RESULTS: Of 60 eligible participants, median age was 34 years and 28% were female. The median pre-ART PEth in the low, moderate, and high PEth groups were <8, 23, and 232 ng/mL, respectively. After starting ART, the CD4 count increased by 13.60 cells/mm3/mo (95% CI: 0.33 to 26.87) with low PEth, 0.93 cells/mm3/mo (95% CI: -6.18 to 8.04) with moderate PEth, and 2.33 cells/mm3/mo (95% CI: -3.44 to 8.09) with high PEth. CONCLUSIONS: Among Russians with HIV, we observed faster CD4 recovery after ART initiation in those with low alcohol consumption compared with those with moderate and high alcohol consumption, as assessed by PEth. This analysis provides further evidence for the possible value of alcohol reduction interventions for people with HIV who are initiating ART.

Soluble CD4 and low molecular weight CD4-mimetic compounds sensitize cells to be killed by anti-HIV cytotoxic immunoconjugates.

IMPORTANCE: HIV infection can be effectively treated to prevent the development of AIDS, but it cannot be cured. We have attached poisons to anti-HIV antibodies to kill the infected cells that persist even after years of effective antiviral therapy. Here we show that the killing of infected cells can be markedly enhanced by the addition of soluble forms of the HIV receptor CD4 or by mimics of CD4.

CD4+CD25+CD127loFOXP3+ cell in food allergy: Does it predict anaphylaxis?

Background: Food allergy (FA), hence the incidence of food anaphylaxis, is a public health problem that has increased in recent years. There are still no biomarkers for patients with FA to predict severe allergic reactions such as anaphylaxis. Objective: There is limited information on whether regulatory T (Treg) cell levels are a biomarker that predicts clinical severity in cases presenting with FA, and which patients are at a greater risk for anaphylaxis. Methods: A total of 70 children were included in the study: 25 who had IgE-mediated cow’s milk protein allergy (CMPA) and presented with non-anaphylactic symptoms (FA/A−), 16 who had IgE-mediated CMPA and presented with anaphylaxis (FA/A+) (a total of 41 FA cases), and a control group consisting of 29 children without FA. The study was conducted by performing CD4+CD25+CD127loFOXP3+ cell flow cytometric analysis during resting at least 2 weeks after the elimination diet to FA subjects. Results: When the FA group was compared with healthy control subjects, CD4+CD25+CD127loFOXP3+ cell rates were found to be significantly lower in the FA group (p < 0.001). When the FA/A− and FA/A+ groups and the control group were compared in terms of CD4+CD25+CD127loFOXP3+ cell ratios, they were significantly lower in the FA/A− and FA/A+ groups compared to the control group (p < 0.001). Conclusions: Although there was no significant difference between the FA/A+ group and the FA/A− group in terms of CD4+CD25+CD127loFOXP3+ cells, our study is important, as it is the first pediatric study we know to investigate whether CD4+CD25+CD127loFOXP3+ cells in FA predict anaphylaxis (AU)

Modelling the impact of CD4 testing on mortality from TB and cryptococcal meningitis among patients with advanced HIV disease in nine countries.

INTRODUCTION: Despite antiretroviral therapy (ART) scale-up among people living with HIV (PLHIV), those with advanced HIV disease (AHD) (defined in adults as CD4 count <200 cells/mm3 or clinical stage 3 or 4), remain at high risk of death from opportunistic infections. The shift from routine baseline CD4 testing towards viral load testing in conjunction with "Test and Treat" has limited AHD identification. METHODS: We used official estimates and existing epidemiological data to project deaths from tuberculosis (TB) and cryptococcal meningitis (CM) among PLHIV-initiating ART with CD4 <200 cells/mm3 , in the absence of select World Health Organization recommended diagnostic or therapeutic protocols for patients with AHD. We modelled the reduction in deaths, based on the performance of screening/diagnostic testing and the coverage and efficacy of treatment/preventive therapies for TB and CM. We compared projected TB and CM deaths in the first year of ART from 2019 to 2024, with and without CD4 testing. The analysis was performed for nine countries: South Africa, Kenya, Lesotho, Mozambique, Nigeria, Uganda, Zambia, Zimbabwe and the Democratic Republic of Congo. RESULTS: The effect of CD4 testing comes through increased identification of AHD and consequent eligibility for protocols for AHD prevention, diagnosis and management; algorithms for CD4 testing avert between 31% and 38% of deaths from TB and CM in the first year of ART. The number of CD4 tests required per death averted varies widely by country from approximately 101 for South Africa to 917 for Kenya. CONCLUSIONS: This analysis supports retaining baseline CD4 testing to avert deaths from TB and CM, the two most deadly opportunistic infections among patients with AHD. However, national programmes will need to weigh the cost of increasing CD4 access against other HIV-related priorities and allocate resources accordingly.

[The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats].

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

A young woman with persistent sore throat, Epstein-Barr virus, lymphadenopathy, and aberrant CD4 + CD7- T-cells.

A young woman with persistent EBV viremia and lymphocytosis had an abnormal CD4- T cell population with aberrant loss of CD7. She had a diagnosis of chronic active EBV (CAEBV), a lymphoproliferative disorder for which she ultimately required allogeneic hematopoietic stem cell transplantation.

The role of CD4+ CD25+ Treg in the mechanism of autoimmune auditory neuropathy in SD rats / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the role of CD4+CD25+regulatory cell (CD4+CD25+Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4+CD25+Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4+CD25+Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4+CD25+Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4+CD25+Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4+CD25+Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4+CD25+Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4+CD25+Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.

Identification of IOMA-class neutralizing antibodies targeting the CD4-binding site on the HIV-1 envelope glycoprotein.

A major goal of current HIV-1 vaccine design efforts is to induce broadly neutralizing antibodies (bNAbs). The VH1-2-derived bNAb IOMA directed to the CD4-binding site of the HIV-1 envelope glycoprotein is of interest because, unlike the better-known VH1-2-derived VRC01-class bNAbs, it does not require a rare short light chain complementarity-determining region 3 (CDRL3). Here, we describe three IOMA-class NAbs, ACS101-103, with up to 37% breadth, that share many characteristics with IOMA, including an average-length CDRL3. Cryo-electron microscopy revealed that ACS101 shares interactions with those observed with other VH1-2 and VH1-46-class bNAbs, but exhibits a unique binding mode to residues in loop D. Analysis of longitudinal sequences from the patient suggests that a transmitter/founder-virus lacking the N276 glycan might have initiated the development of these NAbs. Together these data strengthen the rationale for germline-targeting vaccination strategies to induce IOMA-class bNAbs and provide a wealth of sequence and structural information to support such strategies.

Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer.

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterize Ab1303 and Ab1573, heterologously-neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding is observed only when Env trimers are not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures show that both antibodies recognize the CD4bs on Env trimer with an 'occluded-open' conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation includes outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, does not exhibit V1V2 displacement, 4-stranded gp120 bridging sheet, or co-receptor binding site exposure. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggest an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

Elevated Cerebrospinal Fluid Anti-CD4 Autoantibody Levels in HIV Associate with Neuroinflammation.

The mechanisms of persistent central nervous system (CNS) inflammation in people with HIV (PWH) despite effective antiretroviral therapy (ART) are not fully understood. We have recently shown that plasma anti-CD4 IgGs contribute to poor CD4+ T cell recovery during suppressive ART via antibody-mediated cytotoxicity (ADCC) against CD4+ T cells, and that plasma anti-CD4 IgG levels are associated with worse cognitive performance and specific brain area atrophy. However, the role of anti-CD4 IgGs in neuroinflammation remains unclear. In the current study, plasma and cerebrospinal fluid (CSF) samples from 31 ART-naive and 26 treated, virologically suppressed PWH, along with 16 HIV-seronegative controls, were evaluated for CSF levels of anti-CD4 IgG, white blood cell (WBC) counts, soluble biomarkers of neuroinflammation, and neurofilament light chain (NfL). We found that 37% of the PWH exhibited elevated CSF anti-CD4 IgG levels, but few or none of the PWH were observed with elevated CSF anti-CD4 IgM, anti-CD8 IgG, or anti-double-strand DNA IgG. CSF anti-CD4 IgG levels in PWH were directly correlated with neuroinflammation (WBC counts, neopterin, and markers of myeloid cell activation), but not with CSF NfL levels. Using cells from one immune nonresponder to ART, we generated a pathogenic anti-CD4 monoclonal IgG (JF19) presenting with ADCC activity; JF19 induced the production of soluble CD14 (sCD14) and interleukin-8 (IL-8) in human primary monocyte-derived macrophages via CD4 binding in vitro. This study demonstrates for the first time that elevated CSF anti-CD4 IgG levels present in a subgroup of PWH which may play a role in neuroinflammation in HIV. IMPORTANCE This study reports that an autoantibody presents in the CNS of HIV patients and that its levels in the CSF correlate with some markers of neuroinflammation.

Lymphocytes and sinus histiocytosis in tumor and matched lymph nodes as predictors of survival in non-small-cell lung cancer.

Aim: To analyze immune cell populations in non-small-cell lung cancer (NSCLC) tumors and matched tumor-bearing and non-tumor-bearing lymph nodes (ntbLNs) to predict prognosis. Patients & methods: 71 patients with long-term disease-free survival and 80 patients with relapse within 3 years were included in this study. We used Cox regression to identify factors associated with overall survival (OS) and progression-free survival (PFS). Results: Sinus histiocytosis and tumor-infiltrating lymphocyte density in the tumor were positively associated with PFS and OS. CD4 expression in node 1 (hazard ratio = 0.72; p = 0.02) and node 2 (hazard ratio = 0.91; p = 0.04) ntbLNs were positively correlated with OS and PFS, respectively. Discussion: Immunological markers in ntbLNs could be used to predict survival in NSCLC.

Lay abstract Aim: We analyzed populations of immune cells in non-small-cell lung cancer (NSCLC). In addition, we also investigated lymph nodes from the same patient that contained or did not contain cancer cells. Patients & methods: We included 71 patients whose cancer did not return within 3 years and 80 patients whose cancer did return within 3 years after they underwent surgery to remove their tumors. We used various statistical methods to identify factors that can predict survival. Results: Sinus histiocytosis (a widening of ducts in the lymph nodes due to an increased number of certain cells) and the density of tumor-infiltrating lymphocytes (immune cells that enter the tumor to destroy it) can predict how long patients can survive after surgery or if their tumor will come back quickly. Discussion: Looking at immune cells can help physicians decide which patients need increased follow-up care due to an increased risk for their tumors to return.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies.

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

Effects of Salmonella enterica ser. Enteritidis and Heidelberg on host CD4+CD25+ regulatory T cell suppressive immune responses in chickens.

Poultry infected with Salmonella mount an immune response initially, however the immune responses eventually disappear leading the bird to be a carrier of Salmonella. The hypothesis of this study is that Salmonella infection induces T regulatory cell numbers and cytokine production and suppress host T cells locally in the gut to escape the host immune responses. An experiment was conducted to comparatively analyze the effect of S. enterica ser. Enteritidis (S. Enteritidis) and S. enterica ser. Heidelberg (S. Heidelberg) infection on CD4+CD25+ T regulatory cell properties in chickens. A total of 144 broiler chicks were randomly distributed into three experimental groups of non-infected control, S. Enteritidis infected and S. Heidelberg infected groups. Chickens were orally inoculated with PBS (control) or 5x106 CFU/mL of either S. Enteritidis or S. Heidelberg at 3 d of age. Each group was replicated in six pens with eight chickens per pen. Chickens infected with S. Enteritidis had 6.2, 5.4, and 3.8 log10 CFU/g, and chickens infected with S. Heidelberg had 7.1, 4.8, and 4.1 log10 CFU/g Salmonella in the cecal contents at 4, 11, and 32 dpi, respectively. Both S. Enteritidis and S. Heidelberg were recovered from the liver and spleen 4 dpi. At 4, 11, and 32 dpi, chickens infected with S. Enteritidis and S. Heidelberg had increased CD4+CD25+ cell numbers as well as IL-10 mRNA transcription of CD4+CD25+ cells compared to that in the control group. CD4+CD25+ cells from S. Enteritidis- and S. Heidelberg-infected chickens and restimulated with 1 µg antigen in vitro, had higher (P < 0.05) IL-10 mRNA transcription than the CD4+CD25+ cells from the non-infected controls Though at 4dpi, chickens infected with S. Enteritidis and S. Heidelberg had a significant (P < 0.05) increase in CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, the CD4+CD25- IL-2, IL-1ß, and IFNγ mRNA transcription, were comparable to that in the control group at 11 and 32dpi identifying that the host inflammatory response against Salmonella disappears at 11 dpi. It can be concluded that S. Enteritidis and S. Heidelberg infection at 3 d of age induces a persistent infection through inducing CD4+CD25+ cells and altering the IL-10 mRNA transcription of CD4+CD25+ cell numbers and cytokine production in chickens between 3 to 32 dpi allowing chickens to become asymptomatic carriers of Salmonella after 18 dpi.

Blood transfusion practices affect CD4+ CD25+ FOXP3+ regulatory T cells/T helper-17 cells and the clinical outcome of geriatric patients with hip fracture.

Hip fracture (HF) is common among older individuals and associated with high mortality, poor vitality and functional impairment. HF patients suffer whole body immunological changes and that lead to severe consequences, including immobilization, physical impairment and a high risk of complications. The objective of this study was to decipher the pattern of dynamic immunological changes, especially in two major T cell subsets, CD4+ CD25+ FOXP3+ regulatory T (Treg) cells and T helper-17 (Th17) cells, and their balance, during the hospital stay and to observe whether blood transfusion could influence these cells and clinical patietns' prognosis. In this study, ninety-eight consecutive HF patients were initially enrolled, and finally fifty-one patients qualified for the study, and correlation analysis of their clinical parameters was carried out to predict the meaning of their distribution in clinical practice. Our results showed that the frequency of Tregs gradually decreased, while the frequency of Th17 cells slowly increased in HF patients who received blood transfusion. The Treg frequency was inversely correlated with the level of hemoglobin (Hb), and Th17 cell frequency was positively related to fluctuations in Hb levels in HF patients after trauma. HF patients with a better prognosis and survival time showed decreased a Treg frequency and a decreased Treg/Th17 ratio. Transfusion helped reverse the imbalance in the frequencies of Tregs and Th17 cells and the Treg/Th17 ratio and especially contributed to a better outcome in HF patients with moderate-to-severe anemia. In conclusion, a higher frequency of peripheral blood Tregs and a higher Treg/Th17 ratio may be associated with unfavorable outcomes in HF patients, and blood transfusion may benefit moderate-to-severe HF patients rebalance their immune response.

A Structural Update of Neutralizing Epitopes on the HIV Envelope, a Moving Target.

Antibodies that can neutralize diverse HIV-1 strains develop in ~10-20% of HIV-1 infected individuals, and their elicitation is a goal of vaccine design. Such antibodies can also serve as therapeutics for those who have already been infected with the virus. Structural characterizations of broadly reactive antibodies in complex with the HIV-1 spike indicate that there are a limited number of sites of vulnerability on the spike. Analysis of their structures can help reveal commonalities that would be useful in vaccine design and provide insights on combinations of antibodies that can be used to minimize the incidence of viral resistance mutations. In this review, we give an update on recent structures determined of the spike in complex with broadly neutralizing antibodies in the context of all epitopes on the HIV-1 spike identified to date.

Across Functional Boundaries: Making Nonneutralizing Antibodies To Neutralize HIV-1 and Mediate Fc-Mediated Effector Killing of Infected Cells.

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, "closed" Env trimer present on HIV-1 virions or expressed on HIV-1-infected cells. Antibodies targeting this region are therefore nonneutralizing and their potential as mediators of antibody-dependent cellular cytotoxicity (ADCC) of HIV-1-infected cells diminished by a lack of available binding targets. Here, we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or cluster A specificity to the extracellular domains 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4+ T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties, confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics that are effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening "closed" Env trimers on HIV-1 particles/infected cells to expose the cluster A region and activate ADCC and neutralization against these nonneutralizing targets. IMPORTANCE Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1-infected cells through antibody-dependent cellular cytotoxicity and inactivate HIV-1 virus through neutralization.

Broadly Neutralizing Antibodies for HIV-1 Prevention.

Given the absence of an effective vaccine for protection against HIV-1 infection, passive immunization strategies that utilize potent broadly neutralizing antibodies (bnAbs) to block acquisition of HIV-1 are being rigorously pursued in the clinical setting. bnAbs have demonstrated robust protection in preclinical animal models, and several leading bnAb candidates have shown favorable safety and pharmacokinetic profiles when tested individually or in combinations in early phase human clinical trials. Furthermore, passive administration of bnAbs in HIV-1 infected individuals has resulted in prolonged suppression of viral rebound following interruption of combination antiretroviral therapy, and robust antiviral activity when administered to viremic individuals. Recent results from the first efficacy trials testing repeated intravenous administrations of the anti-CD4 binding site bnAb VRC01 have demonstrated positive proof of concept that bnAb passive immunization can confer protection against HIV-1 infection in humans, but have also highlighted the considerable barriers that remain for such strategies to effectively contribute to control of the epidemic. In this review, we discuss the current status of clinical studies evaluating bnAbs for HIV-1 prevention, highlight lessons learned from the recent Antibody Mediated Prevention (AMP) efficacy trials, and provide an overview of strategies being employed to improve the breadth, potency, and durability of antiviral protection.