Increased Antibody Avidity and Cross-Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Variants by Hyperimmunized Transchromosomic Bovine-Derived Human Immunoglobulins for Treatment of Coronavirus Disease 2019.

Passive antibody immunotherapeutics directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are promising countermeasures for protection and treatment of coronavirus disease 2019 (COVID-19). SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) can impact the clinical efficacy of immunotherapeutics. A fully human polyclonal antibody immunotherapeutic purified from plasma of transchromosomic (Tc) bovines hyperimmunized with SARS-CoV-2 WA-1 spike (SAB-185) is being assessed for efficacy in a phase 2/3 clinical trial when different circulating SARS-CoV-2 variants predominated. We evaluated antibody binding, avidity maturation, and SARS-CoV-2 VOCs/VOIs virus-neutralizing capacity of convalescent plasma compared with different lots of SAB-185 and individual Tc bovine sera sequentially obtained after each vaccination against Alpha, Epsilon, Iota, Gamma, Beta, Kappa, and Delta variants. In contrast to convalescent plasma, sera and SAB-185 derived from hyperimmunized Tc bovines demonstrated higher antibody avidity and more potent cross-neutralizing activity of VOCs/VOIs. Thus, SAB-185 is a potential promising therapeutic candidate for the treatment of patients infected with SARS-CoV-2 variants.

Influence of sample collection tube method, anticoagulant-containing plasma versus serum, on influenza virus hemagglutination inhibition titer and microneutralization titer serological assays.

BACKGROUND: The hemagglutination-inhibition (HAI) assay is a critical component for measurement of immunogenicity in influenza vaccine development. It is unknown if the results can be influenced by sample type and anticoagulants. The purpose of this study was to evaluate the influence of different sample collection methods, in particular different anticoagulants, and choice of plasma or serum, on influenza virus serological assays. METHODS: Blood samples from thirty donors previously immunized against influenza viruses were collected using six different types of blood collection tubes, two of which collect serum and four of which contain various anticoagulants for collecting plasma. Serum: (1) serum separator tubes (SST); and (2) Plus Plastic serum "red-top serum" tubes. Plasma: (3) spray-coated K2 ethylenediaminetetraacetic acid (EDTA) tubes: (4) Sodium Heparin tubes; (5) Citrate tubes with 3.2% sodium citrate solution; and (6) Glass Blood Collection tubes with acid citrate dextrose. Samples were tested against three different influenza viruses (A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012) for hemagglutination inhibition titer and virus neutralization titer via a microneutralization (MN) assay, and data compared to that obtained for standard serum sample collected in SST. RESULTS: HAI and MN titers against type A viruses were within two dilutions compared to SST collection method over 96% of the time irrespective of sample type or anticoagulant. However, HAI titers for type B virus were more variable across different collection methods. EDTA plasma samples were greater than two dilutions higher than SST serum samples 70% (21 of 30 samples) of the time. In contrast, MN titers were within two dilutions over 96% of the time, with the highest deviation noted in acid citrate dextrose plasma samples (3 of 30 samples tested, 10%). CONCLUSIONS: These data provide useful guidelines for sample collection and serology testing when screening: (i) influenza vaccine immunogenicity antibody response; (ii) antibody responses to newly emerging viral strains; and (iii) clinical samples for anti-influenza antibody activity.

Mosquito bite immunization with radiation-attenuated Plasmodium falciparum sporozoites: safety, tolerability, protective efficacy and humoral immunogenicity.

BACKGROUND: In this phase 1 clinical trial, healthy adult, malaria-naïve subjects were immunized with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) by mosquito bite and then underwent controlled human malaria infection (CHMI). The PfRAS model for immunization against malaria had previously induced >90 % sterile protection against homologous CHMI. This study was to further explore the safety, tolerability and protective efficacy of the PfRAS model and to provide biological specimens to characterize protective immune responses and identify protective antigens in support of malaria vaccine development. METHODS: Fifty-seven subjects were screened, 41 enrolled and 30 received at least one immunization. The true-immunized subjects received PfRAS via mosquito bite and the mock-immunized subjects received mosquito bites from irradiated uninfected mosquitoes. Sera and peripheral blood mononuclear cells (PBMCs) were collected before and after PfRAS immunizations. RESULTS: Immunization with PfRAS was generally safe and well tolerated, and repeated immunization via mosquito bite did not appear to increase the risk or severity of AEs. Local adverse events (AEs) of true-immunized and mock-immunized groups consisted of erythaema, papules, swelling, and induration and were consistent with reactions from mosquito bites seen in nature. Two subjects, one true- and one mock-immunized, developed large local reactions that completely resolved, were likely a result of mosquito salivary antigens, and were withdrawn from further participation as a safety precaution. Systemic AEs were generally rare and mild, consisting of headache, myalgia, nausea, and low-grade fevers. Two true-immunized subjects experienced fever, malaise, myalgia, nausea, and rigours approximately 16 h after immunization. These symptoms likely resulted from pre-formed antibodies interacting with mosquito salivary antigens. Ten subjects immunized with PfRAS underwent CHMI and five subjects (50 %) were sterilely protected and there was a significant delay to parasitaemia in the other five subjects. All ten subjects developed humoral immune responses to whole sporozoites and to the circumsporozoite protein prior to CHMI, although the differences between protected and non-protected subjects were not statistically significant for this small sample size. CONCLUSIONS: The protective efficacy of this clinical trial (50 %) was notably less than previously reported (>90 %). This may be related to differences in host genetics or the inherent variability in mosquito biting behavior and numbers of sporozoites injected. Differences in trial procedures, such as the use of leukapheresis prior to CHMI and of a longer interval between the final immunization and CHMI in these subjects compared to earlier trials, may also have reduced protective efficacy. This trial has been retrospectively registered at ISRCTN ID 17372582, May 31, 2016.

No evidence for enhanced disease with human polyclonal SARS-CoV-2 antibody in the ferret model.

Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.

Skin Swabbing for Staphylococcus aureus-Targeting Phages.

INTRODUCTION: Staphylococcus aureus (SA) is a major human bacterial pathogen increasingly refractory to antibiotics. Given the dearth of novel antibiotics in the developmental pipeline, we require concerted efforts at optimizing novel antimicrobial approaches. One promising option is the utilization of bacteriophage (phage) therapy, which has been resurrected as a viable clinical therapeutic. Specifically, an expanded library of phages targeting SA is desired. We surmised that SA-targeting phages would be readily accessible as a major component of the cutaneous microbiome. Specifically, we sought to discern if easily accessible (convenient) and discrete anatomic locations, including the nares, axilla, fingernails, toenails, and web spaces, could provide intact phages via a noninvasive, expedient procedure involving swabbing. METHODS: One hundred subjects participated in systematic skin swab specimen collections. Pooled samples were subject to phage harvesting utilizing the soft agar overlay technique. The approval was secured from the Naval Medical Research Center Institutional Review Board (NMRC 2018.0004 FWA00000152). We utilized the same procedures from known samples containing SA-targeting phages. As another positive control, we employed the same swab and acquired samples from an active wound infection. RESULTS: As anticipated, there were no adverse events, and the procedure was successfully implemented within the projected 10-minute duration. No phages were identified exploiting this methodology. Positive controls from various environmental samples identified SA-targeting phages as did the wound effluent sample. CONCLUSIONS: Skin swabbing at multiple anatomic sites from 100 adults yielded insufficient biomass for phage recovery. The negative results provide helpful information for future phage isolation attempts. The lessons learned on why this study failed to isolate phages can be easily utilized by others. With a desire to increase our SA-targeting phage library in pursuit of future clinical trials, and acknowledging the paucity of these phages accessible via traditional recovery from environmental sources, we will next acquire large volumes of wound effluent from confirmed infected wounds with SA to optimize the biomass for phage recovery.

Human immunoglobulin from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike antigen efficiently neutralizes viral variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.

Hark back: passive immunotherapy for influenza and other serious infections.

The world is experiencing a pandemic of swine-origin influenza virus H1N1. A vaccine to prevent disease is now available, and millions have or will become ill before they can be vaccinated. The ability to use swine-origin influenza virus vaccines as a public health tool has been described as a "race against time." Oseltamivir and related drugs are being used in an effort to reduce morbidity and mortality, but their efficacy for treating severe influenza is suboptimal, and possible wide-spread emergence of oseltamivir-resistant mutants is a concern. Another approach for prevention and treatment of serious influenza is infusion of hyperimmune plasma. The United States has thousands of licensed blood product collection centers that produce millions of liters of plasma licensed by the Food and Drug Administration on an annual basis for the treatment of serious conditions. Immunotherapy using infusion of convalescent plasma (or hyperimmune intravenous immunoglobulin) has been reported to be an effective treatment for severe influenza and other virulent pathogens in animal models and humans. Plasma obtained from those that have recovered or were early recipients of vaccine offers a resource for production of an immediately available and potentially effective therapy at the local, state, and national level. Past, current, and future uses of immunotherapy and current advisory body recommendations for this approach are presented.

Safety and Immunogenicity of a Tetravalent Dengue DNA Vaccine Administered with a Cationic Lipid-Based Adjuvant in a Phase 1 Clinical Trial.

We conducted an open label, dose escalation Phase 1 clinical trial of a tetravalent dengue DNA vaccine (TVDV) formulated in Vaxfectin® to assess safety and immunogenicity. A total of 40 dengue- and flavivirus-naive volunteers received either low-dose (1 mg) TVDV alone (N = 10, group 1), low-dose TVDV (1 mg) formulated in Vaxfectin (N = 10, group 2), or high-dose TVDV (2 mg, group 3) formulated in Vaxfectin® (N = 20). Subjects were immunized intramuscularly with three doses on a 0-, 30-, 90-day schedule and monitored. Blood samples were obtained after each immunization and various time points thereafter to assess anti-dengue antibody and interferon gamma (IFNγ) T-cell immune responses. The most common adverse events (AEs) across all groups included mild to moderate pain and tenderness at the injection site, which typically resolved within 7 days. Common solicited signs and symptoms included fatigue (42.5%), headache (45%), and myalgias (47.5%). There were no serious AEs related to the vaccine or study procedures. No anti-dengue antibody responses were detected in group 1 subjects who received all three immunizations. There were minimal enzyme-linked immunosorbent assay and neutralizing antibody responses among groups 2 and 3 subjects who completed the immunization schedule. By contrast, IFNγ T-cell responses, regardless of serotype specificity, occurred in 70%, 50%, and 79% of subjects in groups 1, 2, and 3, respectively. The largest IFNγ T-cell responses were among group 3 subjects. We conclude that TVDV was safe and well-tolerated and elicited predominately anti-dengue T-cell IFNγ responses in a dose-related fashion.

Meta-analysis: convalescent blood products for Spanish influenza pneumonia: a future H5N1 treatment?

BACKGROUND: Studies from the Spanish influenza era reported that transfusion of influenza-convalescent human blood products reduced mortality in patients with influenza complicated by pneumonia. Treatments for H5N1 influenza are unsatisfactory, and convalescent human plasma containing H5N1 antibodies could be an effective therapy during outbreaks and pandemics. PURPOSE: To determine whether transfusion with influenza-convalescent human blood products reduced the risk for death in patients with Spanish influenza pneumonia. DATA SOURCES: Manual search of English-language journals from 1918 to 1925. Citations from retrieved studies were also searched. STUDY SELECTION: Published English-language studies that had at least 10 patients in the treatment group, used convalescent blood products to treat Spanish influenza pneumonia in a hospital setting, and reported on a control or comparison group. DATA EXTRACTION: Two investigators independently extracted data on study characteristics, outcomes, adverse events, and quality. DATA SYNTHESIS: Eight relevant studies involving 1703 patients were found. Treated patients, who were often selected because of more severe illness, were compared with untreated controls with influenza pneumonia in the same hospital or ward. The overall crude case-fatality rate was 16% (54 of 336) among treated patients and 37% (452 of 1219) among controls. The range of absolute risk differences in mortality between the treatment and control groups was 8% to 26% (pooled risk difference, 21% [95% CI, 15% to 27%]). The overall crude case-fatality rate was 19% (28 of 148) among patients who received early treatment (after <4 days of pneumonia complications) and 59% (49 of 83) among patients who received late treatment (after > or =4 days of pneumonia complications). The range of absolute risk differences in mortality between the early treatment group and the late treatment group was 26% to 50% (pooled risk difference, 41% [CI, 29% to 54%]). Adverse effects included chill reactions and possible exacerbations of symptoms in a few patients. LIMITATIONS: Studies were few and had many methodologic limitations. No study was a blinded, randomized, or placebo-controlled trial. Some pertinent studies may have been missed. CONCLUSIONS: Patients with Spanish influenza pneumonia who received influenza-convalescent human blood products may have experienced a clinically important reduction in the risk for death. Convalescent human H5N1 plasma could be an effective, timely, and widely available treatment that should be studied in clinical trials.