Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

Effective Prophylaxis of COVID-19 in Rhesus Macaques Using a Combination of Two Parenterally-Administered SARS-CoV-2 Neutralizing Antibodies.

SARS-CoV-2 is a respiratory borne pathogenic beta coronavirus that is the source of a worldwide pandemic and the cause of multiple pathologies in man. The rhesus macaque model of COVID-19 was utilized to test the added benefit of combinatory parenteral administration of two high-affinity anti-SARS-CoV-2 monoclonal antibodies (mAbs; C144-LS and C135-LS) expressly developed to neutralize the virus and modified to extend their pharmacokinetics. After completion of kinetics study of mAbs in the primate, combination treatment was administered prophylactically to mucosal viral challenge. Results showed near complete virus neutralization evidenced by no measurable titer in mucosal tissue swabs, muting of cytokine/chemokine response, and lack of any discernable pathologic sequalae. Blocking infection was a dose-related effect, cohorts receiving lower doses (6, 2 mg/kg) resulted in low grade viral infection in various mucosal sites compared to that of a fully protective dose (20 mg/kg). A subset of animals within this cohort whose infectious challenge was delayed 75 days later after mAb administration were still protected from disease. Results indicate this combination mAb effectively blocks development of COVID-19 in the rhesus disease model and accelerates the prospect of clinical studies with this effective antibody combination.

Anatomic Distribution of Intravenously Injected IgG Takes Approximately 1 Week to Achieve Stratum Corneum Saturation in Vaginal Tissues.

i.v. injected Abs have demonstrated protection against simian HIV infection in rhesus macaques, paving the way for the Antibody Mediated Prevention trial in which at-risk individuals for HIV received an i.v. infusion of the HIV broadly neutralizing Ab VRC01. However, the time needed for these Abs to fully distribute and elicit protection at mucosal sites is still unknown. In this study, we interrogate how long it takes for Abs to achieve peak anatomical levels at the vaginal surface following i.v. injection. Fluorescently labeled VRC01 and/or Gamunex-C were i.v. injected into 24 female rhesus macaques (Macaca mulatta) with vaginal tissues and plasma acquired up to 2 wk postinjection. We found that Ab delivery to the vaginal mucosa occurs in two phases. The first phase involves delivery to the submucosa, occurring within 24 h and persisting beyond 1 wk. The second phase is the delivery through the stratified squamous epithelium, needing ∼1 wk to saturate the stratum corneum. This study has important implications for the efficacy of immunoprophylaxis targeting pathogens at the mucosa.

Development of an In Vivo Probe to Track SARS-CoV-2 Infection in Rhesus Macaques.

Infection with the novel coronavirus, SARS-CoV-2, results in pneumonia and other respiratory symptoms as well as pathologies at diverse anatomical sites. An outstanding question is whether these diverse pathologies are due to replication of the virus in these anatomical compartments and how and when the virus reaches those sites. To answer these outstanding questions and study the spatiotemporal dynamics of SARS-CoV-2 infection a method for tracking viral spread in vivo is needed. We developed a novel, fluorescently labeled, antibody-based in vivo probe system using the anti-spike monoclonal antibody CR3022 and demonstrated that it could successfully identify sites of SARS-CoV-2 infection in a rhesus macaque model of COVID-19. Our results showed that the fluorescent signal from our antibody-based probe could differentiate whole lungs of macaques infected for 9 days from those infected for 2 or 3 days. Additionally, the probe signal corroborated the frequency and density of infected cells in individual tissue blocks from infected macaques. These results provide proof of concept for the use of in vivo antibody-based probes to study SARS-CoV-2 infection dynamics in rhesus macaques.

Peer Crowd Identification and Adolescent Health Behaviors: Results From a Statewide Representative Study.

PURPOSE: Peer crowds are macro-level subcultures that share similarities across geographic areas. Over the past decade, dozens of studies have explored the association between adolescent peer crowds and risk behaviors, and how they can inform public health efforts. However, despite the interest, researchers have not yet reported on crowd size and risk levels from a representative sample, making it difficult for practitioners to apply peer crowd science to interventions. The current study reports findings from the first statewide representative sample of adolescent peer crowd identification and health behaviors. METHODS: Weighted data were analyzed from the 2015 Virginia Youth Survey of Health Behaviors ( n = 4,367). Peer crowds were measured via the I-Base Survey™, a photo-based peer crowd survey instrument. Frequencies and confidence intervals of select behaviors including tobacco use, substance use, nutrition, physical activity, and violence were examined to identify high- and low-risk crowds. Logistic regression was used to calculate adjusted odds ratios for each crowd and behavior. RESULTS: Risky behaviors clustered in two peer crowds. Hip Hop crowd identification was associated with substance use, violence, and some depression and suicidal behaviors. Alternative crowd identification was associated with increased risk for some substance use behaviors, depression and suicide, bullying, physical inactivity, and obesity. Mainstream and, to a lesser extent, Popular, identities were associated with decreased risk for most behaviors. CONCLUSIONS: Findings from the first representative study of peer crowds and adolescent behavior identify two high-risk groups, providing critical insights for practitioners seeking to maximize public health interventions by targeting high-risk crowds.

Media Flight Schedules and Seasonality in Relation to Quitline Call Volume.

CONTEXT: Given the high profile, cost, and vulnerability to budget cuts of mass-reach health education campaigns, researchers have cited the need for media buying strategies. OBJECTIVE: The objective of the current study is to fill a gap in the literature by comparing the impact of media flight schedule types in relation to tobacco quitline call volume. DESIGN: The retrospective study was designed to determine whether type of media flight schedule (eg, flighting, continuous, pulsing) impacted number of calls to the Maine Tobacco Helpline, while accounting for number of gross rating points (GRPs), seasonality, holidays, and other factors. SETTING: Maine has 3 designated market areas (DMAs): Portland/Auburn, Bangor, and Presque Isle. MAIN OUTCOME MEASURES: Daily call volume was matched with weekly GRPs. METHODS: A negative binomial regression model was created to examine the relationship among media flight schedules, number of GRPs, and call volume. Gross rating points reflect national networks and local cable TV media buys. A second model examined the association between GRP dose levels and call volume. RESULTS: The number of GRPs was a significant predictor of call volume (P < .001). Weekly number of GRPs within a media flight schedule was the most important indicator for potential effectiveness. Weekly low-dose GRPs were not effective in increasing calls, indicating a minimum threshold. For every 250 GRPs, 29% (or 73) more calls per week were attributed to the media campaigns (P < .001). Weekly quitline call volume was 21% (or 53 calls) lower during the weeks of Christmas, US Thanksgiving, and US Independence Day. CONCLUSION: Type of media flight schedule should be considered in the context of purchasing sufficient weekly, as well as quarterly, rating points to increase tobacco quitline call volume. In addition, our study is the first to quantify and report on lower tobacco quitline call volume during several US holidays.

Regulation of CD4 T cells and their effects on immunopathological inflammation following viral infection.

CD4 T cells help immune responses, but knowledge of how memory CD4 T cells are regulated and how they regulate adaptive immune responses and induce immunopathology is limited. Using adoptive transfer of virus-specific CD4 T cells, we show that naive CD4 T cells undergo substantial expansion following infection, but can induce lethal T helper type 1-driven inflammation. In contrast, memory CD4 T cells exhibit a biased proliferation of T follicular helper cell subsets and were able to improve adaptive immune responses in the context of minimal tissue damage. Our analyses revealed that type I interferon regulates the expansion of primary CD4 T cells, but does not seem to play a critical role in regulating the expansion of secondary CD4 T cells. Strikingly, blockade of type I interferon abrogated lethal inflammation by primary CD4 T cells following viral infection, despite that this treatment increased the numbers of primary CD4 T-cell responses. Altogether, these data demonstrate important aspects of how primary and secondary CD4 T cells are regulated in vivo, and how they contribute to immune protection and immunopathology. These findings are important for rational vaccine design and for improving adoptive T-cell therapies against persistent antigens.

Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption.

BACKGROUND: The discovery of potent and broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) has made passive immunization a potential strategy for the prevention and treatment of HIV infection. We sought to determine whether passive administration of VRC01, a bNAb targeting the HIV CD4-binding site, can safely prevent or delay plasma viral rebound after the discontinuation of antiretroviral therapy (ART). METHODS: We conducted two open-label trials (AIDS Clinical Trials Group [ACTG] A5340 and National Institutes of Health [NIH] 15-I-0140) of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of VRC01 in persons with HIV infection who were undergoing interruption of ART. RESULTS: A total of 24 participants were enrolled, and one serious alcohol-related adverse event occurred. Viral rebound occurred despite plasma VRC01 concentrations greater than 50 µg per milliliter. The median time to rebound was 4 weeks in the A5340 trial and 5.6 weeks in the NIH trial. Study participants were more likely than historical controls to have viral suppression at week 4 (38% vs. 13%, P=0.04 by a two-sided Fisher's exact test in the A5340 trial; and 80% vs. 13%, P<0.001 by a two-sided Fisher's exact test in the NIH trial) but the difference was not significant at week 8. Analyses of virus populations before ART as well as before and after ART interruption showed that VRC01 exerted pressure on rebounding virus, resulting in restriction of recrudescent viruses and selection for preexisting and emerging antibody neutralization-resistant virus. CONCLUSIONS: VRC01 slightly delayed plasma viral rebound in the trial participants, as compared with historical controls, but it did not maintain viral suppression by week 8. In the small number of participants enrolled in these trials, no safety concerns were identified with passive immunization with a single bNAb (VRC01). (Funded by the National Institute of Allergy and Infectious Diseases and others; ACTG A5340 and NIH 15-I-0140 ClinicalTrials.gov numbers, NCT02463227 and NCT02471326 .).

Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection.

Passive immunization with HIV-1-neutralizing monoclonal antibodies (mAbs) is being considered for prevention and treatment of HIV-1 infection. As therapeutic agents, mAbs could be used to suppress active virus replication, maintain suppression induced by antiretroviral therapy (ART), and/or decrease the size of the persistent virus reservoir. We assessed the impact of VRC01, a potent human mAb targeting the HIV-1 CD4 binding site, on ART-treated and untreated HIV-1-infected subjects. Among six ART-treated individuals with undetectable plasma viremia, two infusions of VRC01 did not reduce the peripheral blood cell-associated virus reservoir measured 4 weeks after the second infusion. In contrast, six of eight ART-untreated, viremic subjects infused with a single dose of VRC01 experienced a 1.1 to 1.8 log10 reduction in plasma viremia. The two subjects with minimal responses to VRC01 were found to have predominantly VRC01-resistant virus before treatment. Notably, two subjects with plasma virus load <1000 copies/ml demonstrated virus suppression to undetectable levels for over 20 days until VRC01 levels declined. Among the remaining four subjects with baseline virus loads between 3000 and 30,000 copies, viremia was only partially suppressed by mAb infusion, and we observed strong selection pressure for the outgrowth of less neutralization-sensitive viruses. In summary, a single infusion of mAb VRC01 significantly decreased plasma viremia and preferentially suppressed neutralization-sensitive virus strains. These data demonstrate the virological effect of this neutralizing antibody and highlight the need for combination strategies to maintain virus suppression.

Symmetry Controlled, Genetic Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration Protein.

Viruses use spatial control of constituent proteins as a means of manipulating and evading host immune systems. Similarly, precise spatial control of proteins encapsulated or presented on designed nanoparticles has the potential to biomimetically amplify or shield biological interactions. Previously, we have shown the ability to encapsulate a wide range of guest proteins within the virus-like particle (VLP) from Salmonella typhimurium bacteriophage P22, including antigenic proteins from human pathogens such as influenza. Expanding on this robust encapsulation strategy, we have used the trimeric decoration protein (Dec) from bacteriophage L as a means of controlled exterior presentation on the mature P22 VLP, to which it binds with high affinity. Through genetic fusion to the C-terminus of the Dec protein, either the 17 kDa soluble region of murine CD40L or a minimal peptide designed from the binding region of the "self-marker" CD47 was independently presented on the P22 VLP capsid exterior. Both candidates retained function when presented as a Dec-fusion. Binding of the Dec domain to the P22 capsid was minimally changed across designed constructs, as measured by surface plasmon resonance, demonstrating the broad utility of this presentation strategy. Dec-mediated presentation offers a robust, modular means of decorating the exposed exterior of the P22 capsid in order to further orchestrate responses to internally functionalized VLPs within biological systems.

Prevalence of smoke-free car and home rules in Maine before and after passage of a smoke-free vehicle law, 2007-2010.

INTRODUCTION: This is the first study to examine the prevalence of self-reported smoke-free rules for private cars and homes before and after the passage of a smoke-free vehicle law. METHODS: Data were examined for 13,461 Maine adults aged 18 or older who participated in the Behavioral Risk Factor Surveillance System, a state-based telephone survey covering health topics. Self-reported smoke-free car and home rules, smoking behavior, and demographic variables of age, sex, education, income, and children in household were analyzed for prevalence before and after the state's smoke-free vehicle law was passed. RESULTS: Prevalence of smoke-free car and home rules was significantly higher after Maine's smoke-free vehicle law was passed in the state (P = .004 for car rules and P = .009 for home rules). Variations in smoking rules differed by smoking and demographic variables. People with household incomes of less than $20,000 saw an increase of 14.3% in smoke-free car rules; overall, those with annual incomes of less than $20,000 and those with less than a high school education reported a lower prevalence of smoke-free car rules both before and after the law was passed than did people with higher incomes and higher education levels. The prevalence of smoke-free home rules after the law was implemented was higher among those with 4 or more years of college education than among those with lower levels of education (P = .02). CONCLUSION: The prevalence of smoke-free car and home rules among Maine adults was significantly higher after the passage of a statewide smoke-free vehicle law. This apparent change in smoke-free rule prevalence may be indicative of changing social norms related to the unacceptability of secondhand smoke exposure.

Filtering of MS/MS data for peptide identification.

BACKGROUND: The identification of proteins based on analysis of tandem mass spectrometry (MS/MS) data is a valuable tool that is not fully realized because of the difficulty in carrying out automated analysis of large numbers of spectra. MS/MS spectra consist of peaks that represent each peptide fragment, usually b and y ions, with experimentally determined mass to charge ratios. Whether the strategy employed is database matching or De Novo sequencing, a major obstacle is distinguishing signal from noise. Improved ability to distinguish signal peaks of low intensity from background noise increases the likelihood of correctly identifying the peptide, as valuable information is preserved while extraneous information is not left to mislead. RESULTS: This paper introduces an automated noise filtering method based on the construction of orthogonal polynomials. By subdividing the spectrum into a variable number (3 to 11) of bins, peaks that are considered "noise" are identified at a local level. Using a De Novo sequencing algorithm that we are developing, this filtering method was applied to a published dataset of more than 3000 mass spectra and an original dataset of more than 300 spectra. The samples were peptides from purified known proteins; therefore, the solutions could be compared to the correct sequences and the peaks corresponding to b, y and other fragments of significance could be identified. The same procedure was applied using two other published filtering methods. The ratios of the number of significant peaks that were preserved relative to the total number of peaks in each spectrum were determined. In the event that filtering out too many or too few signal peaks can lead to inaccuracy in sequence determination, the percentage of amino acid residues in the correct positions relative to the total number of amino acid residues in the correct sequence was also calculated for each sequence determined. CONCLUSIONS: The results show that an orthogonal polynomial-based method of distinguishing signal peaks from background in mass spectra preserves a greater portion of signal peaks than compared methods, improving accuracy in sequence determination.

Prescribing physical activity: applying the ACSM protocols for exercise type, intensity, and duration across 3 training frequencies.

When physicians advise patients to attain more physical activity, they usually recommend a walking program. However, in a similar way to no exercise, those embarking on a walking program will typically lose 4 to 6 lb of lean weight and reduce their resting metabolic rate 2% to 3% every decade. These effects may be mitigated by the inclusion of resistance exercise. The American College of Sports Medicine (ACSM) minimum exercise guidelines recommend 20 minutes of aerobic activity 3 days per week, and 1 set (8-12 repetitions) of 8 to 10 resistance exercises to train the major muscle groups 2 days per week. However, large-scale testing of these recommendations in a field setting has been minimal. Men and women between 21 and 80 years (N = 1725) [corrected] participated in a 10-week combined strength and aerobic activity program based on the ACSM protocols for exercise intensity and duration across 3 training frequencies (1, 2, or 3 sessions/week). Across all training frequencies, mean changes included a reduction in body fat of 1.97%, a decrease in fat weight of 1.7 kg, an increase in lean weight of 1.35 kg, a reduction in systolic blood pressure of 3.83 mm Hg, and a reduction in diastolic blood pressure of 1.73 mm Hg. More frequent weekly training sessions were associated with greater improvements in body fat percent, fat weight, and lean weight. Participants responded favorably to the ACSM exercise program with a 91% completion rate and a 95% satisfaction rating. This article presents recommendations for prescribing safe, effective, and time-efficient exercise programs.