Design of an Escherichia coli expressed HIV-1 gp120 fragment immunogen that binds to b12 and induces broad and potent neutralizing antibodies.

b12, one of the few broadly neutralizing antibodies against HIV-1, binds to the CD4 binding site (CD4bs) on the gp120 subunit of HIV-1 Env. Two small fragments of HIV-1 gp120, b121a and b122a, which display about 70% of the b12 epitope and include solubility-enhancing mutations, were designed. Bacterially expressed b121a/b122a were partially folded and could bind b12 but not the CD4bs-directed non-neutralizing antibody b6. Sera from rabbits primed with b121a or b122a protein fragments and boosted with full-length gp120 showed broad neutralizing activity in a TZM-bl assay against a 16-virus panel that included nine Tier 2 and 3 viruses as well as in a five-virus panel previously designed to screen for broad neutralization. Using a mean IC50 cut-off of 50, sera from control rabbits immunized with gp120 alone neutralized only one virus of the 14 non-Tier 1 viruses tested (7%), whereas sera from b121a- and b122a-immunized rabbits neutralized seven (50%) and twelve (86%) viruses, respectively. Serum depletion studies confirmed that neutralization was gp120-directed and that sera from animals immunized with gp120 contained lower amounts of CD4bs-directed antibodies than corresponding sera from animals immunized with b121a/b122a. Competition binding assays with b12 also showed that b121a/2a sera contained significantly higher amounts of antibodies directed toward the CD4 binding site than the gp120 sera. The data demonstrate that it is possible to elicit broadly neutralizing sera against HIV-1 in small animals.

Identification of an HIV-1 clade A envelope that exhibits broad antigenicity and neutralization sensitivity and elicits antibodies targeting three distinct epitopes.

Broadly neutralizing antibodies (bNAbs) PG9 and PG16 were isolated from an International AIDS Vaccine Initiative (IAVI) Protocol G subject infected with human immunodeficiency virus type 1 (HIV-1) clade A. Both antibodies are highly potent and neutralize greater than 70% of viruses tested. We sought to begin immunogen design based on viral sequences from this patient; however, pseudoviruses prepared with 19 envelope sequences from this subject were resistant to neutralization by PG9 and PG16. Therefore, we used a bioinformatics approach to identify closely related viruses that were potentially sensitive to PG9 and PG16. A most-recent common ancestor (MRCA) sequence for the viral envelope (Env) was determined and aligned with 99 subtype A gp160 sequences from the Los Alamos HIV database. Virus BG505.W6M.ENV.C2 (BG505) was found to have the highest degree of homology (73%) to the MRCA sequence. Pseudoviruses prepared with this Env were sensitive to neutralization with a broad panel of bNAbs, including PG9 and PG16. When expressed by 293T cells as soluble gp120, the BG505 monomer bound well to both PG9 and PG16. We further showed that a point mutation (L111A) enabled more efficient production of a stable gp120 monomer that preserves the major neutralization epitopes. Finally, we showed that an adjuvanted formulation of this gp120 protein elicited neutralizing antibodies in rabbits (following a gp120 DNA vaccine prime) and that the antisera competed with bNAbs from 3 classes of nonoverlapping epitopes. Thus, the BG505 Env protein warrants further investigation as an HIV vaccine candidate, as a stand-alone protein, or as a component of a vaccine vector.

A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals.

A protective vaccine against HIV-1 will likely require the elicitation of a broadly neutralizing antibody (bNAb) response. Although the development of an immunogen that elicits such antibodies remains elusive, a proportion of HIV-1 infected individuals evolve broadly neutralizing serum responses over time, demonstrating that the human immune system can recognize and generate NAbs to conserved epitopes on the virus. Understanding the specificities that mediate broad neutralization will provide insight into which epitopes should be targeted for immunogen design and aid in the isolation of broadly neutralizing monoclonal antibodies from these donors. Here, we have used a number of new and established technologies to map the bNAb specificities in the sera of 19 donors who exhibit among the most potent cross-clade serum neutralizing activities observed to date. The results suggest that broad and potent serum neutralization arises in most donors through a limited number of specificities (1-2 per donor). The major targets recognized are an epitope defined by the bNAbs PG9 and PG16 that is associated with conserved regions of the V1, V2 and V3 loops, an epitope overlapping the CD4 binding site and possibly the coreceptor binding site, an epitope sensitive to a loss of the glycan at N332 and distinct from that recognized by the bNAb 2G12 and an epitope sensitive to an I165A substitution. In approximately half of the donors, key N-linked glycans were critical for expression of the epitopes recognized by the bNAb specificities in the sera.

Menstrual Health and Hygiene among Adolescents in the United States.

STUDY OBJECTIVE: Menstrual health in adolescents has been understudied in the United States. We aimed to assess patient and provider perspectives surrounding menstrual health management and screening. DESIGN: Our mixed-methods approach consisted of provider surveys, patient surveys, and patient interviews. SETTING: Participants were recruited from a pediatric gynecology practice or an adolescent medicine clinic at an urban tertiary academic center. PARTICIPANTS: Providers were pediatrics faculty or residents. Patients aged 13-24 years were eligible. INTERVENTION: Participants completed an anonymous survey or semi-structured interview about their experiences with menstrual health. MAIN OUTCOME MEASURES: Descriptive statistics and thematic content analysis were used for quantitative and qualitative data, respectively. Convergent parallel analysis elucidated key findings in both data sets. RESULTS: The provider survey response rate was 65% (69/106); 15% (9/69) of providers consistently asked patients about menstrual products, whereas 44% (27/68) were concerned patients could not afford products. The patient survey response rate was 85% (101/119); 19% (19/101) of respondents reported menstrual hygiene insecurity, 55% (55/101) missed commitments during menses, and 45% (45/101) discussed menstrual products with providers. Fifteen patients were invited for qualitative interviews; 10 were conducted, and thematic saturation occurred. Interviews highlighted the importance of comprehensive early menstrual health education and providers' role in menstrual management. CONCLUSION: Adolescence is a crucial point of entry into health care. Because taboos surrounding menstruation could limit access to health care, menstrual health education must be emphasized. Menstrual health education is provided piecemeal by parents, schools, and providers. Current practice should be reevaluated to consider comprehensive educational approaches in which health care leads.

Nonhuman Primates Are Protected against Marburg Virus Disease by Vaccination with a Vesicular Stomatitis Virus Vector-Based Vaccine Prepared under Conditions to Allow Advancement to Human Clinical Trials.

Vaccines are needed to disrupt or prevent continued outbreaks of filoviruses in humans across Western and Central Africa, including outbreaks of Marburg virus (MARV). As part of a filovirus vaccine product development plan, it is important to investigate dose response early in preclinical development to identify the dose range that may be optimal for safety, immunogenicity, and efficacy, and perhaps demonstrate that using lower doses is feasible, which will improve product access. To determine the efficacious dose range for a manufacturing-ready live recombinant vesicular stomatitis virus vaccine vector (rVSV∆G-MARV-GP) encoding the MARV glycoprotein (GP), a dose-range study was conducted in cynomolgus macaques. Results showed that a single intramuscular injection with as little as 200 plaque-forming units (PFUs) was 100% efficacious against lethality and prevented development of viremia and clinical pathologies associated with MARV Angola infection. Across the vaccine doses tested, there was nearly a 2000-fold range of anti-MARV glycoprotein (GP) serum IgG titers with seroconversion detectable even at the lowest doses. Virus-neutralizing serum antibodies also were detected in animals vaccinated with the higher vaccine doses indicating that vaccination induced functional antibodies, but that the assay was a less sensitive indicator of seroconversion. Collectively, the data indicates that a relatively wide range of anti-GP serum IgG titers are observed in animals that are protected from disease implying that seroconversion is positively associated with efficacy, but that more extensive immunologic analyses on samples collected from our study as well as future preclinical studies will be valuable in identifying additional immune responses correlated with protection that can serve as markers to monitor in human trials needed to generate data that can support vaccine licensure in the future.

A novel, live-attenuated vesicular stomatitis virus vector displaying conformationally intact, functional HIV-1 envelope trimers that elicits potent cellular and humoral responses in mice.

Though vaccination with live-attenuated SIV provides the greatest protection from progressive disease caused by SIV challenge in rhesus macaques, attenuated HIV presents safety concerns as a vaccine; therefore, live viral vectors carrying HIV immunogens must be considered. We have designed a replication-competent vesicular stomatitis virus (VSV) displaying immunogenic HIV-1 Env trimers and attenuating quantities of the native surface glycoprotein (G). The clade B Env immunogen is an Env-VSV G hybrid (EnvG) in which the transmembrane and cytoplasmic tail regions are derived from G. Relocation of the G gene to the 5'terminus of the genome and insertion of EnvG into the natural G position induced a ∼1 log reduction in surface G, significant growth attenuation compared to wild-type, and incorporation of abundant EnvG. Western blot analysis indicated that ∼75% of incorporated EnvG was a mature proteolytically processed form. Flow cytometry showed that surface EnvG bound various conformationally- and trimer-specific antibodies (Abs), and in-vitro growth assays on CD4+CCR5+ cells demonstrated EnvG functionality. Neither intranasal (IN) or intramuscular (IM) administration in mice induced any observable pathology and all regimens tested generated potent Env-specific ELISA titers of 10(4)-10(5), with an IM VSV prime/IN VSV boost regimen eliciting the highest binding and neutralizing Ab titers. Significant quantities of Env-specific CD4+ T cells were also detected, which were augmented as much as 70-fold by priming with IM electroporated plasmids encoding EnvG and IL-12. These data suggest that our novel vector can achieve balanced safety and immunogenicity and should be considered as an HIV vaccine platform.

Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target.

Broadly neutralizing antibodies (bNAbs), which develop over time in some HIV-1-infected individuals, define critical epitopes for HIV vaccine design. Using a systematic approach, we have examined neutralization breadth in the sera of about 1800 HIV-1-infected individuals, primarily infected with non-clade B viruses, and have selected donors for monoclonal antibody (mAb) generation. We then used a high-throughput neutralization screen of antibody-containing culture supernatants from about 30,000 activated memory B cells from a clade A-infected African donor to isolate two potent mAbs that target a broadly neutralizing epitope. This epitope is preferentially expressed on trimeric Envelope protein and spans conserved regions of variable loops of the gp120 subunit. The results provide a framework for the design of new vaccine candidates for the elicitation of bNAb responses.