Validation of a severe acute respiratory syndrome coronavirus 2 microneutralization assay for evaluation of vaccine immunogenicity.

As variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, assessment of vaccine immunogenicity remains a critical factor to support continued vaccination. To this end, an in vitro microneutralization (MN50) assay was validated to quantitate SARS-CoV-2 neutralizing antibodies against prototype and variant strains (Beta, Delta, Omicron BA.1, Omicron BA.5, and XBB.1.5) in human serum. For the prototype strain, the MN50 assay met acceptance criteria for inter-/intra-assay precision, specificity, linearity, and selectivity. The assay was robust against changes to virus/serum incubation time, cell seeding density, virus content per well, cell passage number, and serum interference. Analyte in serum samples was stable up to five freeze/thaw cycles and for up to 12 months of storage at -80 ± 10 °C. Similar results were observed for the variant-adapted MN50 assays. The conversion factor to convert assay result units to WHO international standard units (IU/mL) was determined to be 0.62 for the prototype strain. This MN50 assay will be useful for vaccine immunogenicity analyses in clinical trial samples, enabling assessment of vaccine immunogenicity for ancestral and variant strains as variant-adapted vaccines are developed.

Prevention of antimicrobial prescribing among infants following maternal vaccination against respiratory syncytial virus.

SignificanceStrategies to reduce consumption of antimicrobial drugs are needed to contain the growing burden of antimicrobial resistance. Respiratory syncytial virus (RSV) is a prominent cause of upper and lower respiratory tract infections, as a single agent and in conjunction with bacterial pathogens, and may thus contribute to the burden of both inappropriately treated viral infections and appropriately treated polymicrobial infections involving bacteria. In a double-blind, randomized, placebo-controlled trial, administering an RSV vaccine to pregnant mothers reduced antimicrobial prescribing among their infants by 12.9% over the first 3 mo of life. Our findings implicate RSV as an important contributor to antimicrobial exposure among infants and demonstrate that this exposure is preventable by use of effective maternal vaccines against RSV.

A Phase 2 randomized, observer-blind, placebo-controlled, dose-ranging trial of aluminum-adjuvanted respiratory syncytial virus F particle vaccine formulations in healthy women of childbearing age.

OBJECTIVE: Respiratory syncytial virus (RSV) causes significant morbidity and mortality in infants. We are developing an RSV fusion (F) protein nanoparticle vaccine for immunization of third trimester pregnant women to passively protect infants through transfer of RSV-specific maternal antibodies. The present trial was performed to assess the immunogenicity and safety of several formulations of RSV F vaccine in 1-dose or 2-dose schedules. METHODS: Placebo, or vaccine with 60µg or 120µg RSV F protein and 0.2, 0.4, or 0.8mg aluminum, were administered intramuscularly on Days 0 and 28 to healthy women 18-35years old. Immunogenicity was assessed from Days 0 through 91 based on anti-F IgG and palivizumab-competitive antibody (PCA) by ELISA, and RSV A and B neutralizing antibodies by microneutralization (MN) assay. Solicited adverse events were collected through Day 7 and unsolicited adverse events through Day 91. RESULTS: All formulations were well-tolerated, with no treatment-related serious adverse events. Anti-F IgG and PCA responses were correlated and increased after both doses, while MN increased significantly only after the first dose, then plateaued. The timeliest and most robust antibody responses followed one dose of 120µg RSV F protein and 0.4mg aluminum, but persistence through 91days was modestly (∼25%) superior following two doses of 60µg RSV F protein and 0.8mg aluminum. Western blot analysis showed RSV infections in active vaccinees were reduced by 52% overall (p=0.009 overall) over the Day 0 through 90 period. CONCLUSIONS: RSV F nanoparticle vaccine formulations were well tolerated and immunogenic. The optimal combination of convenience and rapid response for immunization in the third trimester occurred with 120µg RSV F and 0.4mg aluminum, which achieved peak immune responses in 14days and sufficient persistence through 91days to allow for passive transfer of IgG antibodies to the fetus. NCT01960686.

A Randomized, Blinded, Controlled, Dose-Ranging Study of a Respiratory Syncytial Virus Recombinant Fusion (F) Nanoparticle Vaccine in Healthy Women of Childbearing Age.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of infant morbidity and mortality. A recombinant RSV fusion protein nanoparticle vaccine (RSV F vaccine) candidate for maternal immunization was tested for safety and immunogenicity in women of childbearing age. METHODS: Three hundred thirty women (18-35 years) were randomized to receive 1 or 2 doses of RSV F vaccine (60 or 90 µg) with or without aluminum phosphate adjuvant, or placebo at days 0 and 28. Safety was evaluated over 180 days; immunogenicity and RSV infection rates were evaluated over 112 days. RESULTS: All vaccine formulations were well tolerated, without vaccine-related serious adverse events. Anti-F immunoglobulin G antibodies rose 6.5-15.6-fold, with significantly higher levels in 2-dose, adjuvanted regimens at day 56. Palivizumab-competitive antibody levels were undetectable at day 0 but increased up to 325 µg/mL at day 56. A 2.7- and 3.5-fold rise in RSV/A and RSV/B microneutralization antibodies were noted at day 56. Between days 56 and 112, 21% (12/56) of placebo recipients and 11% of vaccinees (26/244) showed evidence of a recent RSV infection (P = .04). CONCLUSIONS: The vaccine appeared safe, immunogenic, and reduced RSV infections. Further development as a vaccine for use in maternal immunization is warranted. CLINICAL TRIALS REGISTRATION: NCT01704365.

Modeling maternal fetal RSV F vaccine induced antibody transfer in guinea pigs.

BACKGROUND: Protection of newborns and young infants against RSV disease via maternal immunization mediated by transplacental transfer of antibodies is under evaluation in third-trimester pregnant women with the RSV recombinant F nanoparticle vaccine (RSV F vaccine). Since the hemichorial placental architecture in guinea pigs and humans is similar, the guinea pig model was employed to assess RSV F vaccine immunogenicity in pregnant sows and to compare RSV-specific maternal antibody levels in their pups. METHODS: Thirty (30) presumptive pregnant guinea pigs were immunized on gestational day 25 and 46 with placebo (PBS), 30µg RSV F, or 30µg RSV F+400µg aluminum phosphate. Sera at delivery/birth (sows/pups) and 15 and 30 days post-partum (pups) were analyzed for the presence of anti-F IgG, palivizumab-competitive antibody (PCA) and RSV/A microneutralization (MN). RESULTS: The rates of pregnancy and stillbirth were similar between controls and vaccinees. The vaccine induced high levels of anti-F IgG, PCA and MN in sows, with the highest levels observed in adjuvanted vaccinees. Placental transfer to pups was proportional to the maternal antibody levels, with concentration effects observed for all immune measures. CONCLUSIONS: The RSV F vaccine was safe and immunogenic in pregnant guinea pigs and supported robust transplacental antibody transfer to their pups. Relative concentration of antibodies in the pups was observed even in the presence of high levels of maternal antibody. Guinea pigs may be an important safety and immunogenicity model for preclinical assessment of candidate vaccines for maternal immunization.

Safety of AS03-adjuvanted inactivated split virion A(H1N1)pdm09 and H5N1 influenza virus vaccines administered to adults: pooled analysis of 28 clinical trials.

Clinical trials have shown that AS03-adjuvanted H5N1 and A(H1N1)pdm09 vaccines are highly immunogenic, although with an increased reactogenicity profile relative to non-adjuvanted vaccines in terms of the incidence of common injection site and systemic adverse events (AEs). We evaluated pooled safety data from 22,521 adults who had received an AS03-adjuvanted H5N1 or A(H1N1)pdm09 influenza or control vaccine with the purpose to identify medically-attended AEs (MAEs), including subsets of serious AEs (SAEs), potentially immune-mediated diseases (pIMDs), and AEs of special interest (AESI), and to explore a potential association of these AEs with the administration of an AS03-adjuvanted influenza vaccine. For participants who had received an AS03-adjuvanted vaccine, the relative risks (RRs) for experiencing a MAE or a SAE compared to control group (participants who had received a non-adjuvanted vaccine or saline placebo) were 1.0 (95% confidence interval [CI]: 0.9; 1.1) and 1.1 (95% CI: 0.9; 1.4), respectively. The overall RRs for experiencing an AESI or a pIMD (AS03-adjuvanted vaccine/control) were 1.2 (95% CI: 0.9; 1.6) and 1.7 (95% CI: 0.8; 3.8), respectively. Thirty-8 participants in the AS03-adjuvanted vaccine group had a pIMD reported after vaccine administration, yielding an incidence rate (IR) of 351.9 (95% CI: 249.1; 483.1) per 100,000 person-years. The estimated IRs in the AS03-adjuvanted vaccine group were greater than the literature reported rates for: facial paresis/VIIth nerve paralysis, celiac disease, thrombocytopenia and ulcerative colitis. These results do not support an association between AS03-adjuvanted H5N1 and A(H1N1)pdm09 vaccines and the AEs collected in the trials included in the analysis.

An insect cell derived respiratory syncytial virus (RSV) F nanoparticle vaccine induces antigenic site II antibodies and protects against RSV challenge in cotton rats by active and passive immunization.

Post-infectious immunity to respiratory syncytial virus (RSV) infection results in limited protection as evidenced by the high rate of infant hospitalization in the face of high titer, maternally derived RSV-specific antibodies. By contrast, RSV fusion (F) glycoprotein antigenic site II humanized monoclonal antibodies, palivizumab and motavizumab, have been shown to reduce RSV-related hospitalization in infants. Immunogenicity and efficacy studies were conducted in cotton rats comparing a recombinant RSV F nanoparticle vaccine with palivizumab and controlled with live RSV virus intranasal immunization and, formalin inactivated RSV vaccine. Active immunization with RSV F nanoparticle vaccine containing an alum adjuvant induced serum levels of palivizumab competing antibody (PCA) greater than passive administration of 15 mg/kg palivizumab (human prophylactic dose) in cotton rats and neutralized RSV-A and RSV-B viruses. Immunization prevented detectable RSV replication in the lungs and, unlike passive administration of palivizumab, in the nasal passage of challenged cotton rats. Histology of lung tissues following RSV challenge showed no enhanced disease in the vaccinated groups in contrast to formalin inactivated 'Lot 100' vaccine. Passive intramuscular administration of RSV F vaccine-induced immune sera one day prior to challenge of cotton rats reduced viral titers by 2 or more log10 virus per gram of lung and nasal tissue and at doses less than palivizumab. A recombinant RSV F nanoparticle vaccine protected lower and upper respiratory tract against both RSV A and B strain infection and induced polyclonal palivizumab competing antibodies similar to but potentially more broadly protective against RSV than palivizumab.

Development of influenza H7N9 virus like particle (VLP) vaccine: homologous A/Anhui/1/2013 (H7N9) protection and heterologous A/chicken/Jalisco/CPA1/2012 (H7N3) cross-protection in vaccinated mice challenged with H7N9 virus.

The recent emergence of severe human illness caused by avian-origin influenza A(H7N9) viruses in China has precipitated a global effort to rapidly develop and test vaccine candidates. To date, non-A(H7N9) H7 subtype influenza vaccine candidates have been poorly immunogenic and difficulties in production of A(H7N9) virus seed strains have been encountered. A candidate recombinant A(H7N9) vaccine consisting of full length, unmodified hemagglutinin (HA) and neuraminidase (NA) from the A/Anhui/1/2013 and the matrix 1 (M1) protein from the A/Indonesia/05/2005 (H5N1) were cloned into a baculovirus vector. Baculovirus infected Spodoptera frugiperda (Sf9) insect cells secreted virus like particles (VLP) composed of HA, NA, and M1 that resemble mature influenza virions. Genetic construction of vaccine from acquisition of an H7N9 genomic sequence to production of A(H7N9) VLP occurred in 26 days. The immunogenicity and efficacy of A/Anhui/1/2013 (H7N9) VLP vaccine administered on days 0 and 14 were evaluated in a lethal wild-type challenge Balb/c mouse model. Control groups included a non-homologous H7 vaccine (A/chicken/Jalisco/CPA1/2012 (H7N3)-VLP), and A/Indonesia/05/2005 (H5N1)-VLP, or placebo. All vaccines were administered with or without ISCOMATRIX. A(H7N9) VLP elicited hemagglutination-inhibition (HAI) antibody titers of ≥ 1:64 against the homologous virus, cross-reactive HAI against the heterologous A(H7N3), and 3- to 4-fold higher HAI responses in corresponding ISCOMATRIX subgroups. Similarly, all doses of H7N9 VLP elicited anti-neuraminidase (NA) antibody, with 3- to 4-fold higher responses measured in the corresponding ISCOMATRIX subgroups. The non-homologous H7 vaccine induced both H7N3 and H7N9 HAI but no N9 anti-NA antibodies. A lethal murine wild-type A/Anhui/1/2013 (H7N9) challenge demonstrated 100% survival of all animals receiving A(H7N9) and A(H7N3) vaccine, versus 0% survival in A(H5N1) vaccine and placebo groups. Together, the data demonstrate that recombinant H7N9 vaccine can be rapidly developed that was immunogenic and efficacious supporting testing in man as a pandemic influenza H7N9 vaccine candidate.

Safety and immunogenicity of 26-valent group a streptococcus vaccine in healthy adult volunteers.

BACKGROUND: Group A streptococcus (GAS) causes illness ranging from uncomplicated pharyngitis to life-threatening necrotizing fasciitis, toxic shock, and rheumatic fever. Attempts to develop an M protein-based vaccine have been hindered by the fact that some M proteins elicit both protective antibodies and antibodies that cross-react with human tissues. New molecular techniques have allowed the previous obstacles to be largely overcome. METHODS: The vaccine is comprised of 4 recombinant proteins adsorbed to aluminum hydroxide that contain N-terminal peptides from streptococcal protective antigen and M proteins of 26 common pharyngitis, invasive, and/or rheumatogenic serotypes. Thirty healthy adult subjects received intramuscular 26-valent GAS vaccine (400 microg) at 0, 1, and 4 months, with clinical and laboratory follow-up for safety and immunogenicity using assays for tissue cross-reactive antibodies, type-specific M antibodies to 27 vaccine antigens, and functional (opsonization) activity of M protein antibodies. RESULTS: The incidence of local reactogenicity was similar to that for other aluminum hydroxide-adsorbed vaccines in adults. No subject developed evidence of rheumatogenicity or nephritogenicity, and no induction of human tissue-reactive antibodies was detected. Overall, 26 of 27 antigenic peptides evoked a >4-fold increase in the geometric mean antibody titer over baseline. The mean log2 fold-increase in serum antibody titer (+/- standard error of the mean) for all 27 antigens was 3.67 +/- 0.21. A significant mean log2 reduction in streptococcal bacterial counts in serum samples obtained after immunization was seen in opsonization assays for all M serotypes. CONCLUSIONS: On the basis of epidemiological data demonstrating that the majority of cases of pharyngitis, necrotizing fasciitis, and other invasive streptococcal infections are caused by a limited number of serotypes, this 26-valent vaccine could have significant impact on the overall burden of streptococcal disease.