Functional Evaluation and Genetic Evolution of Human T-Cell Responses After Vaccination With a Conditionally Replication-Defective Cytomegalovirus Vaccine.

BACKGROUND: Cytomegalovirus (CMV) can cause congenital infection and is the leading cause of nongenetic newborn disabilities. V160, a conditionally replication-defective virus, is an investigational vaccine under evaluation for prevention of congenital CMV. The vaccine was well tolerated and induced both humoral and cellular immunity in CMV-seronegative trial participants. T-cell-mediated immunity is important for immune control of CMV. Here we describe efforts to understand the quality attributes of the T-cell responses induced by vaccination. METHODS: Using multicolor flow cytometry, we analyzed vaccine-induced T cells for memory phenotype, antigen specificity, cytokine profiles, and cytolytic potential. Moreover, antigen-specific T cells were sorted from 4 participants, and next-generation sequencing was used to trace clonal lineage development during the course of vaccination using T-cell receptor ß-chain sequences as identifiers. RESULTS: The results demonstrated that vaccination elicited polyfunctional CD4 and CD8 T cells to 2 dominant antigens, pp65 and IE1, with a predominantly effector phenotype. Analysis of T-cell receptor repertoires showed polyclonal expansion of pp65- and IE1-specific T cells after vaccination. CONCLUSION: V160 induced a genetically diverse and polyfunctional T-cell response and the data support further clinical development of V160 for prevention of CMV infection and congenital transmission. CLINICAL TRIALS REGISTRATION: NCT01986010.

Human monoclonal antibodies isolated from a primary pneumococcal conjugate Vaccinee demonstrates the expansion of an antigen-driven Hypermutated memory B cell response.

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.

Identification and Quantification of a Pneumococcal Cell Wall Polysaccharide by Antibody-Enhanced Chromatography Assay.

Multivalent pneumococcal vaccines have been developed successfully to combat invasive pneumococcal diseases (IPD) and reduce the associated healthcare burden. These vaccines employ pneumococcal capsular polysaccharides (PnPs), either conjugated or unconjugated, as antigens to provide serotype-specific protection. Pneumococcal capsular polysaccharides used for vaccine often contain residual levels of cell wall polysaccharides (C-Ps), which can generate a non-serotype specific immune response and complicate the desired serotype-specific immunity. Therefore, the C-P level in a pneumococcal vaccine needs to be controlled in the vaccine process and the anti C-P responses need to be dialed out in clinical assays. Currently, two types of cell-wall polysaccharide structures have been identified: a mono-phosphocholine substituted cell-wall polysaccharide C-Ps1 and a di-phosphocholine substituted C-Ps2 structure. In our effort to develop a next-generation novel pneumococcal conjugate vaccine (PCV), we have generated a monoclonal antibody (mAb) specific to cell-wall polysaccharide C-Ps2 structure. An antibody-enhanced HPLC assay (AE-HPLC) has been established for serotype-specific quantification of pneumococcal polysaccharides in our lab. With the new anti C-Ps2 mAb, we herein extend the AE-HPLC assay to the quantification and identification of C-Ps2 species in pneumococcal polysaccharides used for vaccines.

Evaluation of luciferase and prefusion-stabilized F protein from respiratory syncytial virus mRNA/LNPs in pre-clinical models using jet delivery compared to needle and syringe.

Described here is the evaluation of a luciferase (luc) and respiratory syncytial virus (RSV) messenger RNA / lipid nanoparticle (mRNA/LNP) vaccine using a Needle-free Injection System, Tropis®, from PharmaJet® (Golden, Colorado USA). Needle-free jet delivery offers an alternative to needle/syringe. To perform this assessment, compatibility studies with Tropis were first performed with a luc mRNA/LNP and compared to needle/syringe. Although minor changes in particle size and encapsulation efficiency were observed when using Tropis on the benchtop, in vitro luciferase activity remained the same. Next, the luc mRNA/LNP was administered to rats intramuscularly using Tropis or needle/syringe and tracking of the injection and distribution was performed. Lastly, an mRNA encoding a prefusion-stabilized F protein from RSV was delivered intramuscularly using both Tropis and needle/syringe at 1 and 5 mcg mRNA. An equivalent IgG response was observed using both Tropis and needle/syringe. The cell mediated immune (CMI) response was also evaluated, and responses to RSV-F were detected from animals immunized with needle/syringe at all dose levels, and from the animals immunized with Tropis in the 5 and 25 ug groups. These results indicated that delivery of mRNA/LNPs with Tropis is a potential means of administration and an alternative to needle/syringe.

Pneumococcal Vaccine Breakthrough and Failure in Infants and Children: A Narrative Review.

Globally, Streptococcus pneumoniae is a leading cause of vaccine-preventable morbidity and mortality in infants and children. In recent decades, large-scale pediatric immunization programs have substantially reduced the incidence of invasive pneumococcal disease. Despite this, residual vaccine-type pneumococcal disease remains in the form of vaccine breakthrough and vaccine failure. This targeted literature review aims to discuss aspects of vaccine breakthrough and failure in infants and children, including disease epidemiology, clinical presentation, risk factors, vaccination schedules, vaccine serotypes, correlates of protection, comorbidities, disease surveillance, and potential implications for future vaccine development.

Characterization of humoral and cell-mediated immunity induced by mRNA vaccines expressing influenza hemagglutinin stem and nucleoprotein in mice and nonhuman primates.

In response to immune pressure, influenza viruses evolve, producing drifted variants capable of escaping immune recognition. One strategy for inducing a broad-spectrum immune response capable of recognizing multiple antigenically diverse strains is to target conserved proteins or protein domains. To that end, we assessed the efficacy and immunogenicity of mRNA vaccines encoding either the conserved stem domain of a group 1 hemagglutinin (HA), a group 2 nucleoprotein (NP), or a combination of the two antigens in mice, as well as evaluated immunogenicity in naïve and influenza seropositive nonhuman primates (NHPs). HA stem-immunized animals developed a robust anti-stem antibody binding titer, and serum antibodies recognized antigenically distinct group 1 HA proteins. These antibodies showed little to no neutralizing activity in vitro but were active in an assay measuring induction of antibody-dependent cellular cytotoxicity. HA-directed cell-mediated immunity was weak following HA stem mRNA vaccination; however, robust CD4 and CD8 T cell responses were detected in both mice and NHPs after immunization with mRNA vaccines encoding NP. Both HA stem and NP mRNA vaccines partially protected mice from morbidity following lethal influenza virus challenge, and superior efficacy against two different H1N1 strains was observed when the antigens were combined. In vivo T cell depletion suggested that anti-NP cell-mediated immunity contributed to protection in the mouse model. Taken together, these data show that mRNA vaccines encoding conserved influenza antigens, like HA stem and NP in combination, induce broadly reactive humoral responses as well as cell-mediated immunity in mice and NHPs, providing protection against homologous and heterologous influenza infection in mice.

A phase I study to evaluate safety, pharmacokinetics, and pharmacodynamics of respiratory syncytial virus neutralizing monoclonal antibody MK-1654 in healthy Japanese adults.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among all infants worldwide and remains a significant cause of morbidity and mortality. To address this unmet medical need, MK-1654, a half-life extended RSV neutralizing monoclonal antibody, is in clinical development for the prevention of RSV disease in infants. This was a phase I, randomized, placebo-controlled, single-site, double-blind trial of MK-1654 in 44 healthy Japanese adults. The safety, tolerability, pharmacokinetics, antidrug antibodies (ADAs), and serum neutralizing antibody (SNA) titers against RSV were evaluated for 1 year after a single intramuscular (i.m.) or intravenous (i.v.) dose of MK-1654 or placebo in five groups (100 mg i.m., 300 mg i.m., 300 mg i.v., 1000 mg i.v., or placebo). MK-1654 was generally well-tolerated in Japanese adults. There were no serious drug-related adverse events (AEs) reported in any MK-1654 recipient and no discontinuations due to any AEs in the study. The half-life of MK-1654 ranged from 76 to 91 days across dosing groups. Estimated bioavailability was 86% for 100 mg i.m. and 77% for 300 mg i.m. One participant out of 33 (3.0%) developed detectable ADA with no apparent associated AEs. The RSV SNA titers increased in a dose-dependent manner among participants who received MK-1654. These data support the development of MK-1654 for use in Japanese infants.

A phase I randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and immunogenicity of a live-attenuated quadrivalent dengue vaccine in flavivirus-naïve and flavivirus-experienced healthy adults.

Dengue (DENV) is a mosquito-borne virus with four serotypes causing substantial morbidity in tropical and subtropical areas worldwide. V181 is an investigational, live, attenuated, quadrivalent dengue vaccine. In this phase 1 double-blind, placebo-controlled study, the safety, tolerability, and immunogenicity of V181 in baseline flavivirus-naïve (BFN) and flavivirus-experienced (BFE) healthy adults were evaluated in two formulations: TV003 and TV005. TV005 contains a 10-fold higher DENV2 level than TV003. Two-hundred adults were randomized 2:2:1 to receive TV003, TV005, or placebo on Days 1 and 180. Immunogenicity against the 4 DENV serotypes was measured using a Virus Reduction Neutralization Test (VRNT60) after each vaccination and out to 1 year after the second dose. There were no discontinuations due to adverse events (AE) or serious vaccine-related AEs in the study. Most common AEs after TV003 or TV005 were headache, rash, fatigue, and myalgia. Tri- or tetravalent vaccine-viremia was detected in 63.9% and 25.6% of BFN TV003 and TV005 participants, respectively, post-dose 1 (PD1). Tri- or tetravalent dengue VRNT60 seropositivity was demonstrated in 92.6% of BFN TV003, 74.2% of BFN TV005, and 100% of BFE TV003 and TV005 participants PD1. Increases in VRNT60 GMTs were observed after the first vaccination with TV003 and TV005 in both flavivirus subgroups for all dengue serotypes, and minimal increases were measured PD2. GMTs in the TV003 and TV005 BFE and BFN groups remained above the respective baselines and placebo through 1-year PD2. These data support further development of V181 as a single-dose vaccine for the prevention of dengue disease.

Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

Efficacy of multivalent adenovirus-based vaccine against simian immunodeficiency virus challenge.

The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01(+)/B*17(-) Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01(+) cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env approximately Gag/Pol > Gag approximately Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.

PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

AIDS clinical trials group 5197: a placebo-controlled trial of immunization of HIV-1-infected persons with a replication-deficient adenovirus type 5 vaccine expressing the HIV-1 core protein.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1)-specific cellular immunity contributes to the control of HIV-1 replication. HIV-1-infected volunteers who were receiving antiretroviral therapy were given a replication-defective adenovirus type 5 HIV-1 gag vaccine in a randomized, blinded therapeutic vaccination study. METHODS: HIV-1-infected vaccine or placebo recipients underwent analytical treatment interruption (ATI) for 16 weeks. The log(10) HIV-1 RNA load at the ATI set point and the time-averaged area under the curve served as co-primary end points. Immune responses were measured by intracellular cytokine staining and carboxyfluorescein succinimidyl ester dye dilution. RESULTS: Vaccine benefit trends were seen for both primary end points, but they did not reach a prespecified significance level of P < or = 25. The estimated shifts in the time-averaged area under the curve and the ATI set point were 0.24 (P=.04, unadjusted) and 0.26 (P=.07, unadjusted) log(10) copies lower, respectively, in the vaccine arm than in the placebo arm. HIV-1 gag-specific CD4(+) cells producing interferon-gamma were an immunologic correlate of viral control. CONCLUSION: The vaccine was generally safe and well tolerated. Despite a trend favoring viral suppression among vaccine recipients, differences in HIV-1 RNA levels did not meet the prespecified level of significance. Induction of HIV-1 gag-specific CD4 cells correlated with control of viral replication in vivo. Future immunogenicity studies should require a substantially higher immunogenicity threshold before an ATI is contemplated.

Development of a microneutralization assay for HSV-2.

BACKGROUND: Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development. METHODS: We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC's isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis. RESULTS: We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R2 = 0.8250) and presence (R2 = 0.7075) of complement. CONCLUSIONS: We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT.

A phase 1, randomized, placebo-controlled study to evaluate the safety and immunogenicity of an mRNA-based RSV prefusion F protein vaccine in healthy younger and older adults.

Respiratory Syncytial Virus (RSV) causes lower respiratory tract infections that can be severe and sometimes fatal. The risk for severe RSV infection is highest in infants and older adults. A safe and effective RSV vaccine for older adults represents a serious unmet medical need due to higher morbidity and mortality in this age group. In this randomized, partially double-blind, placebo-controlled, phase 1 dose-escalation study, we evaluated the safety, tolerability and immunogenicity of an investigational messenger ribonucleic acid (mRNA) vaccine encoding the RSV fusion protein (F) stabilized in the prefusion conformation. The study was conducted in healthy younger adults (ages ≥18 and ≤49 years) and healthy older adults (ages ≥60 and ≤79 years). Participants received mRNA-1777 (V171) or placebo as a single intramuscular dose. For each dose level, three sentinel participants were administered open-label mRNA-1777 (V171). Seventy-two younger adults were randomized and administered 25, 100, or 200 µg mRNA-1777 (V171) or placebo, and 107 older adults were randomized and administered 25, 100, 200 or 300 µg mRNA-1777 (V171) or placebo. Primary objectives were safety and tolerability and secondary objectives included humoral and cell-mediated immunogenicity. All dose levels of mRNA-1777 (V171) were generally well tolerated and no serious adverse events related to the vaccine were reported. Immunization with mRNA-1777 (V171) elicited a humoral immune response as measured by increases in RSV neutralizing antibody titers, serum antibody titers to RSV prefusion F protein, D25 competing antibody titers to RSV prefusion F protein, and cell-mediated immune responses to RSV-F peptides.

A Phase 1 Randomized, Double-Blind, Placebo-Controlled Trial to Assess the Safety, Tolerability, and Pharmacokinetics of a Respiratory Syncytial Virus Neutralizing Monoclonal Antibody MK-1654 in Healthy Adults.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and related morbidity and mortality in infants. Passive immunization with an RSV-neutralizing antibody can provide rapid protection to this vulnerable population. Proof-of-concept for this approach has been demonstrated by palivizumab; however, the use of this antibody is generally restricted to the highest-risk infants due to monthly dosing requirements and its cost. To address the large unmet medical need for most infants, we are evaluating MK-1654, a fully human RSV-neutralizing antibody with half-life extending mutations targeting site IV of the fusion protein. In this 2-part, placebo-controlled, double-blind, first-in-human study, 152 healthy adults were randomized 3:1 to receive a single dose of MK-1654 or placebo in 5 cohorts (100 or 300 mg as an intramuscular dose or 300, 1000, or 3000 mg as an intravenous dose). Safety, pharmacokinetics, antidrug antibodies, and RSV serum-neutralizing antibody titers were evaluated through 1 year. MK-1654 serum concentrations increased proportionally with dose and resulted in corresponding elevations in RSV serum-neutralizing antibody titers. The antibody displayed a half-life of 73 to 88 days and an estimated bioavailability of 69% at the 300-mg dose. The overall safety profile of MK-1654 was similar to placebo, and treatment-emergent antidrug antibodies were low (2.6%) with no associated adverse events. These data support the continued development of MK-1654 for the prevention of RSV disease in infants.

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection.

The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.

DNA gag/adenovirus type 5 (Ad5) gag and Ad5 gag/Ad5 gag vaccines induce distinct T-cell response profiles.

Results from Merck's phase II adenovirus type 5 (Ad5) gag/pol/nef test-of-concept trial showed that the vaccine lacked efficacy against human immunodeficiency virus (HIV) infection in a high-risk population. Among the many questions to be explored following this outcome are whether (i) the Ad5 vaccine induced the quality of T-cell responses necessary for efficacy and (ii) the lack of efficacy in the Ad5 vaccine can be generalized to other vector approaches intended to induce HIV type 1 (HIV-1)-specific T-cell responses. Here we present a comprehensive evaluation of the T-cell response profiles from cohorts of clinical trial subjects who received the HIV CAM-1 gag insert delivered by either a regimen with DNA priming followed by Ad5 boosting (n = 50) or a homologous Ad5/Ad5 prime-boost regimen (n = 70). The samples were tested using a statistically qualified nine-color intracellular cytokine staining assay measuring interleukin-2 (IL-2), tumor necrosis factor alpha, macrophage inflammatory protein 1beta, and gamma interferon production and expression of CD107a. Both vaccine regimens induced CD4(+) and CD8(+) HIV gag-specific T-cell responses which variably expressed several intracellular markers. Several trends were observed in which the frequencies of HIV-1-specific CD4(+) T cells and IL-2 production from antigen-specific CD8(+) T cells in the DNA/Ad5 cohort were more pronounced than in the Ad5/Ad5 cohort. Implications of these results for future vaccine development will be discussed.

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.

A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

The persistence of HIV reservoirs, including latently infected, resting CD4+ T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4+ T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4+ T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV+ or SIV+ subjects, we found that most of the circulating memory CD32+ CD4+ T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a TH2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4+ T cells in peripheral blood or lymphoid tissue; isolated CD32+ resting CD4+ T cells accounted for less than 3% of the total HIV DNA in CD4+ T cells. Cell-associated HIV DNA and RNA loads in CD4+ T cells positively correlated with the frequency of CD32+ CD69+ CD4+ T cells but not with CD32 expression on resting CD4+ T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4+ T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4+ T cells enriched for transcriptionally active HIV after long-term ART.