Primary HIV infection features colonic damage and neutrophil inflammation yet containment of microbial translocation.

INTRODUCTION: Impairment of the gastrointestinal barrier leads to microbial translocation and peripheral immune activation, which are linked to disease progression. Data in the setting of primary HIV/SIV infection suggest that gut barrier damage is one of the first events of the pathogenic cascade, preceding mucosal immune dysfunction and microbial translocation. We assessed gut structure and immunity as well as microbial translocation in acutely and chronically-infected, combination antiretroviral therapy (cART)-naive individuals. METHODS: Fifteen people with primary HIV infection (P-HIV) and 13 with chronic HIV infection (C-HIV) c-ART-naive participants were cross-sectionally studied. Gut biopsies were analysed in terms of gut reservoirs (total, integrated and unintegrated HIV DNA); tight junction proteins (E-cadherin, Zonula Occludens-1), CD4 + expression, neutrophil myeloperoxidase (histochemical staining); collagen deposition (Masson staining). Flow cytometry was used to assess γδ T-cell frequency (CD3 + panγδ+Vδ1+/Vδ2+). In plasma, we measured microbial translocation (LPS, sCD14, EndoCAb) and gut barrier function (I-FABP) markers (ELISA). RESULTS: P-HIV displayed significantly higher tissue HIV DNA, yet neutrophil infiltration and collagen deposition in the gut were similar in the two groups. In contrast, microbial translocation markers were significantly lower in P-HIV compared with C-HIV. A trend to higher mucosal E-cadherin, and gut γδ T-cells was also observed in P-HIV. CONCLUSION: Early HIV infection features higher HIV DNA in the gut, yet comparable mucosal alterations to those observed in chronic infection. In contrast, microbial translocation is contained in primary HIV infection, likely because of a partial preservation of E-cadherin and mucosal immune subsets, namely γδ T-cells.

Rapid monitoring of SARS-CoV-2 variants of concern through high-resolution melt analysis.

The current global pandemic of COVID-19 is characterized by waves of infection due to the emergence of new SARS-CoV-2 variants carrying mutations on the Spike (S) protein gene. Since autumn 2020 many Variants of Concern (VOC) have been reported: Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, Omicron/B.1.1.529, and sublineages. Surveillance of genomic variants is currently based on whole-genome sequencing (WGS) of viral genomes on a random fraction of samples positive to molecular tests. WGS involves high costs, extended analysis time, specialized staff, and expensive instruments compared to a PCR-based test. To rapidly identify the VOCs in positive samples, six assays based on real-time PCR and high-resolution melting (HRM) were designed on the S gene and applied to 120 oro/nasopharyngeal swab samples collected from October 2020 to June 2022 (106 positive and 14 negative samples). Overall, the assays showed 100% specificity and sensitivity compared with commercial PCR tests for COVID-19. Moreover, 104 samples out of 106 (98.1%) were correctly identified as follows: 8 Wuhan (wild type), 12 Alpha, 23 Delta, 46 Omicron BA.1/BA.1.1, 15 Omicron BA.2/BA.4/BA.5. With our lab equipment, about 10 samples can be processed every 3 h at the cost of less than € 10 ($ 10.60) per sample, including RNA extraction. The implementation of this approach could help local epidemiological surveillance and clinical decision-making.

Comparing Heterologous and Homologous COVID-19 Vaccination: A Longitudinal Study of Antibody Decay.

The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.

SARS-CoV-2 RNA Detection on Environmental Surfaces in a University Setting of Central Italy.

The transmission of SARS-CoV-2 occurs through direct contact (person to person) and indirect contact by means of objects and surfaces contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. In this study, we evaluated the presence of SARS-CoV-2 RNA on surfaces made of different materials located in university environments frequented by students and staff involved in academy activity during the fourth pandemic wave (December 2021). A total of 189 environmental samples were collected from classrooms, the library, computer room, gym and common areas and subjected to real-time PCR assay to evaluate the presence of SARS-CoV-2 RNA by amplification of the RNA-dependent RNA polymerase (RdRp) gene. All samples gave a valid result for Internal Process Control and nine (4.8%) tested very low positive for SARS-CoV-2 RNA amplification with a median Ct value of 39.44 [IQR: 37.31-42.66] (≤1 copy of viral genome). Our results show that, despite the prevention measures implemented, the presence of infected subjects cannot be excluded, as evidenced by the recovery of SARS-CoV-2 RNA from surfaces. The monitoring of environmental SARS-CoV-2 RNA could support public health prevention strategies in the academic and school world.

Persistence of Unintegrated HIV DNA Associates With Ongoing NK Cell Activation and CD34+DNAM-1brightCXCR4+ Precursor Turnover in Vertically Infected Patients Despite Successful Antiretroviral Treatment.

The quantification of proviral DNA is raising interest in view of clinical management and functional HIV eradication. Measures of all unintegrated HIV DNA (uDNA) forms in infected reservoir cells provides information on recent replication events that is not found from other proviral DNA assays. To evaluate its actual relevance in a cohort of perinatally-infected adult HIV patients (PHIV), we studied how peripheral blood mononuclear cell uDNA levels correlated with total HIV DNA (tDNA) and with overall replication or innate immune control parameters including NK cell activation/exhaustion and lymphoid turnover. Twenty-two PHIV were included, with successfully controlled HIV (HIV RNA <50 copies/mL) on combined antiretroviral therapy for mean of 8.7 ± 3.9 years. uDNA accounted for 16 [5.2-83.5] copies/µg and was strongly correlated with tDNA (ρ=0.700, p=0.001). Flow cytometric analysis of peripheral NK cells showed that CD69 expression was directly correlated uDNA (p=0.0412), but not with tDNA. Interestingly, CD56-CD16+NK cells which include newly described inflammatory precursors and terminally differentiated cells were directly correlated with uDNA levels (p<0.001), but not with tDNA, and an inverse association was observed between the proportion of NKG2D+ NK cells and uDNA (ρ=-0.548, p=0.015). In addition, CD34+DNAM-1brightCXCR4+ inflammatory precursor frequency correlated directly with uDNA levels (ρ=0.579, p=0.0075). The frequencies of CD56-CD16+ and CD34+DNAM-1brightCXCR4+ cells maintained association with uDNA levels in a multivariable analysis (p=0.045 and p=0.168, respectively). Thus, control of HIV-1 reservoir in aviremic patients on ART is an active process associated with continuous NK cell intervention and turnover, even after many years of treatment. Quantification of linear and circular uDNA provides relevant information on the requirement for ongoing innate immune control in addition to ART, on recent replication history and may help stratify patients for functional HIV eradication protocols with targeted options.

Evaluation of Two-Month Antibody Levels after Heterologous ChAdOx1-S/BNT162b2 Vaccination Compared to Homologous ChAdOx1-S or BNT162b2 Vaccination.

We evaluated the post-vaccination humoral response of three real-world cohorts. Vaccinated subjects primed with ChAdOx1-S and boosted with BNT162b2 mRNA vaccine were compared to homologous dosing (BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S). Serum samples were collected two months after vaccination from a total of 1248 subjects. The results showed that the heterologous vaccine schedule induced a significantly higher humoral response followed by homologous BNT162b2/BNT162b2 and ChAdOx1-S/ChAdOx1-S vaccines (p < 0.0001). Moreover, analyzing factors (i.e., vaccine schedule, sex, age, BMI, smoking, diabetes, cardiovascular diseases, respiratory tract diseases, COVID-19 diagnosis, vaccine side effects) influencing the IgG anti-S response, we found that only the type of vaccine affected the antibody titer (p < 0.0001). Only mild vaccine reactions resolved within few days (40% of subjects) and no severe side effects for either homologous groups or the heterologous group were reported. Our data support the use of heterologous vaccination as an effective and safe alternative to increase humoral immunity against COVID-19.

Prevalence of SARS-CoV-2 infection in the workplace: results of a year of investigation in the Marche Nord companies.

BACKGROUND AND AIM: The world of work has been profoundly affected by the COVID-19 pandemic since workplace activities involving close contact with coworkers and customers can lead to transmission of SARS-CoV-2 and compromise continuity of operations of workplaces. The Covid-Lab of University of Urbino and the Confindustria Pesaro Urbino association signed an agreement to support the Marche Nord companies in the adoption of anti-contagion safety protocols. METHODS: Antibodies detection was performed using a rapid immunochromatographic test. Total RNA from nasopharyngeal swab was subjected to a real-time RT-PCR multiplex for the detection of RdRp specific gene and E gene of the SARS-CoV-2, and the internal control (human RNase P). RESULTS: Between May 2020 and Apr 2021, over 10,000 rapid serological tests had been carried out on workers of 35 companies and in 5% of cases IgG or IgM were found (519). All the 519 swabs gave a valid result (RNAse P Ct≤40) with 105 positive results (20%) for SARS-CoV-2 with a Ct value ≤45. Overall, only 1% of samples resulted positive for viral RNA (105/10000). CONCLUSIONS: The University of Urbino set up a rapid-response (within 24 h, generally <6 h) diagnostic centre for SARS-CoV-2 detection (Covid-Lab) allowing the companies to activate the optimal safety path to ensure the health and safety of workers in the workplace. Our observations during this first year of activity, highlight that in the workplace, the infection does not seem to spread if precautionary measures are followed and only 1% (1 worker out of 100) tested positive for the SARS-CoV-2 virus.

Defining rules governing recognition and Fc-mediated effector functions to the HIV-1 co-receptor binding site.

BACKGROUND: The binding of HIV-1 Envelope glycoproteins (Env) to host receptor CD4 exposes vulnerable conserved epitopes within the co-receptor binding site (CoRBS) which are required for the engagement of either CCR5 or CXCR4 co-receptor to allow HIV-1 entry. Antibodies against this region have been implicated in the protection against HIV acquisition in non-human primate (NHP) challenge studies and found to act synergistically with antibodies of other specificities to deliver effective Fc-mediated effector function against HIV-1-infected cells. Here, we describe the structure and function of N12-i2, an antibody isolated from an HIV-1-infected individual, and show how the unique structural features of this antibody allow for its effective Env recognition and Fc-mediated effector function. RESULTS: N12-i2 binds within the CoRBS utilizing two adjacent sulfo-tyrosines (TYS) for binding, one of which binds to a previously unknown TYS binding pocket formed by gp120 residues of high sequence conservation among HIV-1 strains. Structural alignment with gp120 in complex with the co-receptor CCR5 indicates that the new pocket corresponds to TYS at position 15 of CCR5. In addition, structure-function analysis of N12-i2 and other CoRBS-specific antibodies indicates a link between modes of antibody binding within the CoRBS and Fc-mediated effector activities. The efficiency of antibody-dependent cellular cytotoxicity (ADCC) correlated with both the level of antibody binding and the mode of antibody attachment to the epitope region, specifically with the way the Fc region was oriented relative to the target cell surface. Antibodies with poor Fc access mediated the poorest ADCC whereas those with their Fc region readily accessible for interaction with effector cells mediated the most potent ADCC. CONCLUSION: Our data identify a previously unknown binding site for TYS within the assembled CoRBS of the HIV-1 virus. In addition, our combined structural-modeling-functional analyses provide new insights into mechanisms of Fc-effector function of antibodies against HIV-1, in particular, how antibody binding to Env antigen affects the efficiency of ADCC response.

A comparative analysis of unintegrated HIV-1 DNA measurement as a potential biomarker of the cellular reservoir in the blood of patients controlling and non-controlling viral replication.

BACKGROUND: The persistence of HIV-1 in reservoir cells is one of the major obstacles to eradicating the virus in infected individuals receiving combination antiretroviral therapy (ART). HIV-1 persists in infected cells as a stable integrated genome and more labile unintegrated DNA (uDNA), which includes linear, 1-LTR and 2-LTR circular DNA. 2-LTR circle DNA, although less abundant, is considered a surrogate marker of recent infection events and is currently used instead of the other unintegrated species as a diagnostic tool. This pilot study aimed to investigate how to best achieve the measurement of uDNA. METHODS: A comparative analysis of two qPCR-based methods (U-assay and 2-LTR assay) was performed on the blood of 12 ART-naïve, 14 viremic and 29 aviremic On-ART patients and 20 untreated spontaneous controllers (HIC), sampled at a single time point. RESULTS: The U-assay, which quantified all unintegrated DNA species, showed greater sensitivity than the 2-LTR assay (up to 75%, p < 0.0001), especially in viremic subjects, in whom other forms, in addition to 2-LTR circles, may also accumulate due to active viral replication. Indeed, in aviremic On-ART samples, the U-assay unexpectedly measured uDNA in a higher proportion of samples (76%, 22/29) than the 2-LTR assay (41%, 12/29), (p = 0.0164). A trend towards lower uDNA levels was observed in aviremic vs viremic On-ART patients, reaching significance when we combined aviremic On-ART and HIC (controllers) vs Off-ART and viremic On-ART subjects (non-controllers) (p = 0.0003), whereas 2-LTR circle levels remained constant (p ≥ 0.2174). These data were supported by the high correlation found between uDNA and total DNA (r = 0.69, p < 0.001). CONCLUSIONS: The great advantage of the U-assay is that, unlike the 2-LTR assay, it allows the accurate evaluation of the totality of uDNA that can still be measured even during successful ART when plasma viremia is below the cut-off of common clinical tests (< 50 copies/mL) and 2-LTR circles are more likely to be under the quantification limit. UDNA measurement in blood cells may be used as a biomarker to reveal a so far hidden or underestimated viral reservoir. The potential clinical relevance of uDNA quantification may lead to improvements in diagnostic methods to support clinical strategies.

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding.

Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

A MUC16 IgG Binding Activity Selects for a Restricted Subset of IgG Enriched for Certain Simian Immunodeficiency Virus Epitope Specificities.

We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from patients chronically infected with human immunodeficiency virus (HIV), who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque simian immunodeficiency virus SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcγR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcγR engagement and subsequent ADCC effector function, as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcγRs. Testing of the SIV antigen specificity of IgG from SIV-infected macaques revealed that the MUC16-eluted antibodies were enriched for certain specific epitopes, including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and the subsequent association with MUC16 suggests that the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.IMPORTANCE Understanding how antibodies are distributed in the mucosal environment is valuable for developing a vaccine to block HIV infection. Here, we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16, and interestingly, these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model, which is shown here to have MUC16-targeted antigen responses.

Reduction of Immune Activation and Partial Recovery of Staphylococcal Enterotoxin B-Induced Cytokine Production After Switching to an Integrase Strand Transfer Inhibitor-Containing Regimen: Results from an Observational Cohort Study.

BACKGROUND AND OBJECTIVE: Despite integrase strand transfer inhibitor (INSTI)-containing regimens now being considered a preferred option for both initial therapy and switching strategies in virologically suppressed patients, their effects on lymphocyte phenotypes and functions in the course of effective combination antiretroviral therapy (cART) are still unclear. Thus, we investigated the effect of a 24-week elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (EVG/c/FTC/TDF) regimen on the T cell compartment and HIV reservoirs in HIV-infected patients switching from a suppressive protease inhibitor-based regimen. METHODS: Thirty HIV-positive patients receiving suppressive tenofovir disoproxil fumarate/emtricitabine (TDF + FTC) (for a median of 5 years) in association with either darunavir/ritonavir (DVR/r) (47%) or atazanavir/ritonavir (ATV/r) (53%) were followed up for 24 weeks after switching to EVG/c/FTC/TDF. At baseline (week 0 [W0]) and after 12 (W12) and 24 (W24) weeks we analyzed HLA-DR (human leukocyte antigen-DR isotype)/CD38/Ki67/CCR7 (C-C chemokine receptor type 7)/CD45RA/CD127/PD-1 (programmed cell death-1) on CD4/CD8, interferon (IFN)-γ/interleukin (IL)-2 after HIV/Staphylococcal enterotoxin B (SEB) exposure (flow cytometry); total, integrated, and unintegrated HIV-DNA; and residual low-level HIV viremia (quantitative polymerase chain reaction [qPCR]). RESULTS: While EVG/c/FTC/TDF introduction resulted in a stable CD4+ and CD8+ count, residual low-level HIV-RNA viremia, and HIV reservoirs, we observed a significant reduction in both activated CD4+ (p = 0.016) and CD8+ (p = 0.048) T cells, coupled with an increase in IL-2 and IFN-γ release by CD4+ and CD8+ effector memory T cells, and a decrease in cytokine production by terminally differentiated CD8+ T cells following SEB exposure. Furthermore, the magnitude of the reduction of activated HLA-DR + CD38 + CD8+ T cells (r = - 0.63, p = 0.014) inversely correlates with the amount of total HIV-DNA at W24. CONCLUSIONS: Our data show a favorable effect of EVG/c/FTC/TDF switch to preserve immune activation-driven damage to T cell homeostasis, restore the multifunctional properties of effector T cells, and possibly contain cell-associated HIV viral burden in already virologically suppressed patients.

Deciphering Fc-mediated Antiviral Antibody Functions in Animal Models.

Longstanding discordances and enigmas persist as to the specificities and other properties of antibodies (Abs) most effective in preventing or limiting many viral infections in mammals; in turn, failure to decipher key complexities has added to headwinds for both Ab-based therapeutic approaches and rational vaccine design. More recently, experimental approaches have emerged-and continue to emerge-for discerning the functional role of Ab structure, especially the Fc portion of antibody, in combating viral infections in vivo. A wide range of in vitro measures of antibody activity, from neutralization to antibody-dependent cell mediated cytotoxicity (ADCC)-each of these terms representing only an operational notion defined by the particulars of a given assay-are poised for assignment of both relevance and reliability in forecasting outcomes of infection. Of the several emergent technical opportunities for clarity, attention here is drawn to three realms: the increasing array of known modifications that can be engineered into Abs to affect their in vivo activities; the improvement of murine models involving knockouts and knock-ins of host genes including Fc receptors; and the development of additional virological design tools to differentiate Abs that act primarily by inhibiting viral entry from antibodies that mainly target viral antigens (Ags) on cell surfaces. To illustrate some of the opportunities with either zoonotic (emerging, spillover) or ancient human-adapted viruses, we draw examples from a wide range of viruses that affect humans.

Concurrent Exposure of Neutralizing and Non-neutralizing Epitopes on a Single HIV-1 Envelope Structure.

The trimeric envelope spikes on the HIV-1 virus surface initiate infection and comprise key targets for antiviral humoral responses. Circulating virions variably present intact envelope spikes, which react with neutralizing antibodies; and altered envelope structures, which bind non-neutralizing antibodies. Once bound, either type of antibody can enable humoral effector mechanisms with the potential to control HIV-1 infection in vivo. However, it is not clear how the presentation of neutralizing vs. non-neutralizing epitopes defines distinct virus populations and/or envelope structures on single particles. Here we used single-virion fluorescence correlation spectroscopy (FCS), fluorescence resonance energy transfer (FRET), and two-color coincidence FCS approaches to examine whether neutralizing and non-neutralizing antibodies are presented by the same envelope structure. Given the spatial requirements for donor-acceptor energy transfer (≤10 nm), FRET signals generated by paired neutralizing and non-neutralizing fluorescent Fabs should occur via proximal binding to the same target antigen. Fluorescent-labeled Fabs of the neutralizing anti-gp120 antibodies 2G12 and b12 were combined with Fabs of the non-neutralizing anti-gp41 antibody F240, previously thought to mainly bind gp41 "stumps." We find that both 2G12-F240 and/or b12-F240 Fab combinations generate FRET signals on multiple types of virions in solution. FRET efficiencies position the neutralizing and non-neutralizing epitopes between 7.1 and 7.8 nm apart; potentially fitting within the spatial dimensions of a single trimer-derived structure. Further, the frequency of FRET detection suggests that at least one of such structures occurs on the majority of particles in a virus population. Thus, there is frequent, overlapping presentation of non-neutralizing and neutralizing epitope on freely circulating HIV-1 surfaces. Such information provides a broader perspective of how anti-HIV humoral immunity interfaces with circulating virions.

Induction of Fc-Mediated Effector Functions Against a Stabilized Inner Domain of HIV-1 gp120 Designed to Selectively Harbor the A32 Epitope Region.

Recent clinical trials and studies using nonhuman primates (NHPs) suggest that antibody-mediated protection against HIV-1 will require α-HIV envelope humoral immunity beyond direct neutralization to include Fc-receptor (FcR) mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC). There is also strong evidence indicating that the most potent ADCC response in humans is directed toward transitional non-neutralizing epitopes associated with the gp41-interactive face of gp120, particularly those within the first and second constant (C1-C2) region (A32-like epitopes). These epitopes were shown to be major targets of ADCC responses during natural infection and have been implicated in vaccine-induced protective immunity. Here we describe the immunogenicity of ID2, an immunogen consisting of the inner domain of the clade A/E 93TH057 HIV-1 gp120 expressed independently of the outer domain (OD) and stabilized in the CD4-bound conformation to harbor conformational A32 region epitopes within a minimal structural unit of HIV-1 Env. ID2 induced A32-specific antibody responses in BALB/c mice when injected alone or in the presence of the adjuvants Alum or GLA-SE. Low α-ID2 titers were detected in mice immunized with ID2 alone whereas robust responses were observed with ID2 plus adjuvant, with the greatest ID2 and A32-specific titers observed in the GLA-SE group. Only sera from groups immunized in the presence of GLA-SE were capable of mediating significant ADCC using NKr cells sensitized with recombinant BaL gp120 as targets and human PBMCs as effectors. A neutralization response to a tier 2 virus was not observed. Altogether, our studies demonstrate that ID2 is highly immunogenic and elicits A32-specific ADCC responses in an animal host. The ID2 immunogen has significant translational value as it can be used in challenge studies to evaluate the role of non-neutralizing antibodies directed at the A32 subregion in HIV-1 protection.

Continued Decay of HIV Proviral DNA Upon Vaccination With HIV-1 Tat of Subjects on Long-Term ART: An 8-Year Follow-Up Study.

Introduction: Tat, a key HIV virulence protein, has been targeted for the development of a therapeutic vaccine aimed at cART intensification. Results from phase II clinical trials in Italy (ISS T-002) and South Africa (ISS T-003) indicated that Tat vaccination promotes increases of CD4+ T-cells and return to immune homeostasis while reducing the virus reservoir in chronically cART-treated patients. Here we present data of 92 vaccinees (59% of total vaccinees) enrolled in the ISS T-002 8-year extended follow-up study (ISS T-002 EF-UP, ClinicalTrials.gov NCT02118168). Results: Anti-Tat antibodies (Abs) induced upon vaccination persisted for the entire follow-up in 34/92 (37%) vaccinees, particularly when all 3 Ab classes (A/G/M) were present (66% of vaccinees), as most frequently observed with Tat 30 µg regimens. CD4+ T cells increased above study-entry levels reaching a stable plateau at year 5 post-vaccination, with the highest increase (165 cells/µL) in the Tat 30 µg, 3 × regimen. CD4+ T-cell increase occurred even in subjects with CD4+ nadir ≤ 250 cells/uL and in poor immunological responders and was associated with a concomitant increase of the CD4+/CD8+ T-cell ratio, a prognostic marker of morbidity/mortality inversely related to HIV reservoir size. Proviral DNA load decreased over time, with a half-life of 2 years and an estimated 90% reduction at year 8 in the Tat 30 µg, 3 × group. In multivariate analysis the kinetic and amplitude of both CD4+ T-cell increase and proviral DNA reduction were fastest and highest in subjects with all 3 anti-Tat Ab classes and in the 30 µg, 3 × group, irrespective of drug regimens (NNRTI/NRTI vs. PI). HIV proviral DNA changes from baseline were inversely related to CD4+/CD8+ T-cell ratio and CD4+ T-cell changes, and directly related to the changes of CD8+ T cells. Further, HIV DNA decay kinetics were inversely related to the frequency and levels of intermittent viremia. Finally, Tat vaccination was similarly effective irrespective of the individual immunological status or HIV reservoir size at study entry. Conclusions: Tat immunization induces progressive immune restoration and reduction of virus reservoirs above levels reached with long-term cART, and may represent an optimal vaccine candidate for cART intensification toward HIV reservoirs depletion, functional cure, and eradication strategies.

Different kinetics of viral replication and DNA integration in the main HIV-1 cellular reservoirs in the presence and absence of integrase inhibitors.

To compare the kinetics of integration, p24 production and equilibrium of the different HIV-DNA forms in human primary cells in the presence/absence of integrase-inhibitors (INIs) in vitro. Monocyte-derived-macrophages (MDMs), CD4+ T-cells and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1 in the presence/absence of raltegravir and dolutegravir. HIV-DNA levels and p24 production were measured by qPCR and ELISA assays, respectively. In the absence of INIs, levels of HIV-DNA forms were initially very low, with an increase in the integration process starting at 3 dpi. HIV-DNA increased more slowly in MDMs than it did in CD4+ T-cells and PMBCs peaking at 21 dpi with a mean of 1580 (±890) and 615 (±37) copies/103 cells for proviral and unintegrated HIV-DNA, and 455,972 (±213,255) pg/mL of p24 at the same time point. In CD4+ T-cells the proviral HIV-DNA increased together with unintegrated HIV-DNA peaking at 7 dpi (583 ±â€¯261 and 338 ±â€¯254 copies/103 cells) when the p24 was 218,000 (±75,600) pg/mL. A similar trend was observed in PBMCs (494 ±â€¯361 and 350 ±â€¯123 copies/103 cells for proviral and unintegrated HIV-DNA, and p24 production of 149,400 ±â€¯131,800 pg/mL). Both INIs inhibited viral replication and integration in all the cell types that were tested, especially starting at 3 dpi. However, a small but measurable amount of HIV-DNA (<5 copies/103 cells) was still observed in treated-MDMs up to 30 dpi. In conclusion, our study showed differences in HIV-DNA kinetic integration between CD4+ T-cells and MDMs, which could explain the divergent kinetics of viral-replication. Both INIs inhibited HIV-1 integration and replication with no difference found between CD4+ T-cells and MDMs. However, residual HIV-DNA remained detectable up to 30 dpi in INI-treated MDMs although complete inhibition of HIV replication was achieved. The clinical significance of this minor DNA persistence deserves further investigation considering the role of macrophages as reservoirs.

Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

Application of area scaling analysis to identify natural killer cell and monocyte involvement in the GranToxiLux antibody dependent cell-mediated cytotoxicity assay.

Several different assay methodologies have been described for the evaluation of HIV or SIV-specific antibody-dependent cell-mediated cytotoxicity (ADCC). Commonly used assays measure ADCC by evaluating effector cell functions, or by detecting elimination of target cells. Signaling through Fc receptors, cellular activation, cytotoxic granule exocytosis, or accumulation of cytolytic and immune signaling factors have been used to evaluate ADCC at the level of the effector cells. Alternatively, assays that measure killing or loss of target cells provide a direct assessment of the specific killing activity of antibodies capable of ADCC. Thus, each of these two distinct types of assays provides information on only one of the critical components of an ADCC event; either the effector cells involved, or the resulting effect on the target cell. We have developed a simple modification of our previously described high-throughput ADCC GranToxiLux (GTL) assay that uses area scaling analysis (ASA) to facilitate simultaneous quantification of ADCC activity at the target cell level, and assessment of the contribution of natural killer cells and monocytes to the total observed ADCC activity when whole human peripheral blood mononuclear cells are used as a source of effector cells. The modified analysis method requires no additional reagents and can, therefore, be easily included in prospective studies. Moreover, ASA can also often be applied to pre-existing ADCC-GTL datasets. Thus, incorporation of ASA to the ADCC-GTL assay provides an ancillary assessment of the ability of natural and vaccine-induced antibodies to recruit natural killer cells as well as monocytes against HIV or SIV; or to any other field of research for which this assay is applied. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.