Measured Neutralizing Antibody Titers Research Articles

Neutralizing activity of intravenous immune globulin products against enterovirus D68 strains isolated in Japan

BackgroundEnterovirus D68 (EV-D68), belonging to Enterovirus D, is a unique human enterovirus mainly associated with common respiratory diseases. However, EV-D68 can cause severe respiratory diseases, and EV-D68 endemic is epidemiologically linked to current global epidemic of acute flaccid myelitis.MethodsIn this study, we measured neutralizing antibody titers against six clinical EV-D68 isolates in nine intravenous immune globulin (IVIG) products commercially available in Japan to assess their potential as therapeutic options for severe EV-D68 infection.ResultsSeven IVIG products manufactured from Japanese donors contained high neutralizing antibody titers (IC50 = 0.22–85.01 µg/mL) against all six EV-D68 strains. Apparent differences in neutralizing titers among the six EV-D68 strains were observed for all IVIG products derived from Japanese and non-Japanese blood donors.ConclusionsHigh levels of EV-D68–neutralizing antibodies in IVIG products manufactured from Japanese donors suggest that anti-EV-D68 antibodies are maintained in the Japanese donor population similarly as found in foreign blood donors. Apparent differences in neutralizing antibody titers against the six EV-D68 strains suggest distinct antigenicity among the strains used in this study regardless of the genetic similarity of EV-D68.

Determining the Time of Booster Dose Based on the Half-Life and Neutralization Titers against SARS-CoV-2 Variants of Concern in Fully Vaccinated Individuals.

Although mRNA-based COVID-19 vaccines reduce the risk of severe disease, hospitalization and death, vaccine effectiveness (VE) against infection and disease from variants of concern (VOC) wanes over time. Neutralizing antibodies (NAb) are surrogates of protection and are enhanced by a booster dose, but their kinetics and durability remain understudied. Current recommendation of a booster dose does not consider the existing NAb in each individual. Here, we investigated 50% neutralization (NT50) titers against VOC among COVID-19-naive participants receiving the Moderna (n = 26) or Pfizer (n = 25) vaccine for up to 7 months following the second dose, and determined their half-lives. We found that the time it took for NT50 titers to decline to 24, equivalent to 50% inhibitory dilution of 10 international units/mL, was longer in the Moderna (325/324/235/274 days for the D614G/alpha/beta/delta variants) group than in the Pfizer (253/252/174/226 days) group, which may account for the slower decline in VE of the Moderna vaccine observed in real-world settings and supports our hypothesis that measuring the NT50 titers against VOC, together with information on NAb half-lives, can be used to dictate the time of booster vaccination. Our study provides a framework to determine the optimal time of a booster dose against VOC at the individual level. In response to future VOC with high morbidity and mortality, a quick evaluation of NAb half-lives using longitudinal serum samples from clinical trials or research programs of different primary-series vaccinations and/or one or two boosters could provide references for determining the time of booster in different individuals. IMPORTANCE Despite improved understanding of the biology of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the evolutionary trajectory of the virus is uncertain, and the concern of future antigenically distinct variants remains. Current recommendations for a COVID-19 vaccine booster dose are primarily based on neutralization capacity, effectiveness against circulating variants of concern (VOC), and other host factors. We hypothesized that measuring neutralizing antibody titers against SARS-CoV-2 VOC together with half-life information can be used to dictate the time of booster vaccination. Through detailed analysis of neutralizing antibodies against VOC among COVID-19-naive vaccinees receiving either of two mRNA vaccines, we found that the time it took for 50% neutralization titers to decline to a reference level of protection was longer in the Moderna than in the Pfizer group, which supports our hypothesis. In response to future VOC with potentially high morbidity and mortality, our proof-of-concept study provides a framework to determine the optimal time of a booster dose at the individual level.

Neutralizing antibody levels associated with injectable and aerosolized Ad5-nCoV boosters and BA.2 infection

BackgroundSeveral COVID-19 vaccines are in widespread use in China. Few data exist on comparative immunogenicity of different COVID-19 vaccines given as booster doses. We aimed to assess neutralizing antibody levels raised by injectable and inhaled aerosolized recombinant adenovirus type 5 (Ad5)-vectored COVID-19 vaccine as a heterologous booster after an inactivated COVID-19 vaccine two-dose primary series.MethodsUsing an open-label prospective cohort design, we recruited 136 individuals who had received inactivated vaccine primary series followed by either injectable or inhaled Ad5-vectored vaccine and measured neutralizing antibody titers against ancestral SARS-CoV-2 virus and Omicron BA.1 and BA.5 variants. We also measured neutralizing antibody levels in convalescent sera from 39 patients who recovered from Omicron BA.2 infection.ResultsSix months after primary series vaccination, neutralizing immunity against ancestral SARS-CoV-2 was low and neutralizing immunity against Omicron (B.1.1.529) was lower. Boosting with Ad5-vectored vaccines induced a high immune response against ancestral SARS-CoV-2. Neutralizing responses against Omicron BA.5 were ≥ 80% lower than against ancestral SARS-CoV-2 in sera from prime-boost subjects and in convalescent sera from survivors of Omicron BA.2 infection. Inhaled aerosolized Ad5-vectored vaccine was associated with greater neutralizing titers than injectable Ad5-vectored vaccine against ancestral and Omicron SARS-CoV-2 variants.ConclusionsThese findings support the current strategy of heterologous boosting with injectable or inhaled Ad5-vectored SARS-CoV-2 vaccination of individuals primed with inactivated COVID-19 vaccine.

Functional immune responses against SARS-CoV-2 variants of concern after fourth COVID-19 vaccine dose or infection in patients with blood cancer

Patients with blood cancer continue to have a greater risk of inadequate immune responses following three COVID-19 vaccine doses and risk of severe COVID-19 disease. In the context of the CAPTURE study (NCT03226886), we report immune responses in 80 patients with blood cancer who received a fourth dose of BNT162b2. We measured neutralizing antibody titers (NAbTs) using a live virus microneutralization assay against wild-type (WT), Delta, and Omicron BA.1 and BA.2 and T cell responses against WT and Omicron BA.1 using an activation-induced marker (AIM) assay. The proportion of patients with detectable NAb titers and T cell responses after the fourth vaccine dose increased compared with that after the third vaccine dose. Patients who received B cell-depleting therapies within the 12 months before vaccination have the greatest risk of not having detectable NAbT. In addition, we report immune responses in 57 patients with breakthrough infections after vaccination.

Retention of Neutralizing Response against SARS-CoV-2 Omicron Variant in Sputnik V-Vaccinated Individuals.

The new Omicron variant of SARS-CoV-2, first identified in November 2021, is rapidly spreading all around the world. Omicron has become the dominant variant of SARS-CoV-2. There are many ongoing studies evaluating the effectiveness of existing vaccines. Studies on the neutralizing activity of vaccinated sera against the Omicron variant are currently being carried out in many laboratories. In this study, we have shown the neutralizing activity of sera against the SARS-CoV-2 Omicron variant compared to the reference Wuhan D614G variant in individuals vaccinated with two doses of Sputnik V up to 6 months after vaccination and in individuals who experienced SARS-CoV-2 infection either before or after vaccination. As a control to our study we also measured neutralizing antibody titers in individuals vaccinated with two doses of BNT162b2. The decrease in NtAb titers to the Omicron variant was 8.1-fold for the group of Sputnik V-vaccinated individuals. When the samples were stratified for the time period after vaccination, a 7.6-fold or 8.8-fold decrease in NtAb titers was noticed after up to 3 and 3-to-6 months after vaccination. We observed a 6.7- and 5-fold decrease in Sputnik V-vaccinated individuals experiencing asymptomatic or symptomatic infection, respectively. These results highlight the observation that the decrease in NtAb to the SARS-CoV-2 Omicron variant compared to the Wuhan variant occurs for different COVID-19 vaccines in use, with some showing no neutralization at all, confirming the necessity of a third booster vaccination.

MO920: SARS-COV-2 Neutralizing Antibody Response to Booster Vaccination in Patients on Hemodialysis

BACKGROUND AND AIMSSARS-CoV-2 antibody titers after two doses of vaccination decrease over time. Hemodialysis patients are especially vulnerable to COVID-19 as they are immunocompromised, putting them at higher risk of infection and poorer response to vaccines. Therefore, administrating the third dose (‘booster’) in these patients is key to reduce COVID-19 infections and prevent severe illness. Dialysis patients were among the first group of patients who received booster vaccinations. To study the humoral response to the third injection in this group, we collected serum from 33 patients on hemodialysis and measured neutralizing antibody titers against SARS-CoV-2 before and after their booster doses.METHODPatients were recruited from a dialysis center in New York City, NY from June to September 2021. Data on COVID-19 vaccination and demographics were collected upon enrollment. Blood samples were taken after enrollment. SARS-CoV-2 neutralization antibodies were assayed using the GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (Cat#L00847-A). Corresponding neutralizing antibody titers are presented as Unit/mL (U/mL).RESULTSA total of 33 in-center hemodialysis patients who had received three doses of vaccination were studied. Patients had a mean age of 61 years, 23 (70%) were male. Out of these, 31 (94%) patients received three doses of mRNA-1273 (Moderna), and two patients received the BNT162b2 (Pfizer BioNTech) vaccine. A total of 138 serum samples were analyzed (ranging from 156 days before to 85 days after the booster). Figure 1 shows the antibody titer distribution of all samples in these 33 patients. Each color indicates an individual patient. Each patient has up to 12 data points before and after the booster. The mean neutralizing antibody titers of all 48 data points pre-booster is 29.291 U/mL (range: 228–188.600). Seven days post-booster, the mean neutralizing antibody titer is 73.088 U/mL (range: 12.401–254.504). Mean titer is 169.826 U/mL (range: 17.830–375.046) at 14–28 days post-booster. After the peak time, we observe a decline of the titers. At 72–85 days, the mean titer is 72.179 (range: 33.702–204.382).We fitted a nonparametric mixed effects model with an adaptive spline and a random intercept for each subject to neutralizing antibody titers on the log10 scale. The estimate of the mean trajectory and its 95% confidence interval are shown in Fig. 2. The estimated peak time is 18.2 days with a 95% confidence interval (0–27.7).CONCLUSIONOur results suggest that hemodialysis patients have a strong humoral response to booster vaccination. Neutralizing antibody titers peak at 18 days post-booster and wane to an average of 42% of peak value after 10–12 weeks.FIGURE 1:Time-course of neutralizing antibody titers before and after booster vaccination. The colors identify individual hemodialysis patients.FIGURE 2:A nonparametric mixed effects model with an adaptive spline and a random intercept for each subject to neutralizing antibody titers. The red line indicates the average titer, and the gray area indicates the 95% confidence interval. The circles are means across all data points.

Mumps virus-specific immune response outcomes and sex-based differences in a cohort of healthy adolescents

Despite high levels of MMR-II usage in the US, mumps outbreaks continue to occur. Evidence suggests that mumps vaccine-induced humoral immunity wanes over time. Relatively few studies have examined cell-mediated immunity or reported on sex-based differences. To better understand sex-based differences in the immune response to mumps vaccine, we measured neutralizing antibody titers and mumps-specific cytokine/chemokine responses in a cohort of 748 adolescents and young adults after two doses of MMR vaccine. We observed significantly higher neutralizing antibody titers in females than in males (120.8 IU/mL, 98.7 IU/mL, p = 0.038) but significantly higher secretion levels of MIP-1α, MIP-1β, TNFα, IL-6, IFNγ, and IL-1β in males compared to females. These data demonstrate that sex influences mumps-specific humoral and cell-mediated immune response outcomes, a phenomenon that should be considered during efforts to improve vaccines and prevent future outbreaks.

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

ABSTRACTThe global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.

H5N1 influenza vaccine induces a less robust neutralizing antibody response than seasonal trivalent and H7N9 influenza vaccines

Conventional inactivated avian influenza vaccines have performed poorly in past vaccine trials, leading to the hypothesis that they are less immunogenic than seasonal influenza vaccines. We tested this hypothesis by comparing the immunogenicity of the H5N1 and H7N9 vaccines (avian influenza vaccines) to a seasonal trivalent inactivated influenza vaccine in naïve ferrets, administered with or without the adjuvants MF59 or AS03. Vaccine immunogenicity was assessed by measuring neutralizing antibody titers against hemagglutinin and neuraminidase and by hemagglutinin -specific IgG levels. Two doses of unadjuvanted vaccines induced low or no HA-specific IgG responses and hemagglutination-inhibiting titers. Adjuvanted vaccines induced comparable IgG-titers, but poorer neutralizing antibody titers for the H5 vaccine. All adjuvanted vaccines elicited detectable anti- neuraminidase -antibodies with the exception of the H5N1 vaccine, likely due to the low amounts of neuraminidase in the vaccine. Overall, the H5N1 vaccine had poorer capacity to induce neutralizing antibodies, but not HA-specific IgG, compared to H7N9 or trivalent inactivated influenza vaccine.

Role of Maternal Antibodies in Infants with Severe Diseases Related to Human Parechovirus Type 3.

Human parechovirus type 3 (HPeV3) is an emerging pathogen that causes sepsis and meningoencephalitis in young infants. To test the hypothesis that maternal antibodies can protect this population, we measured neutralizing antibody titers (NATs) to HPeV3 and other genotypes (HPeV1 and HPeV6) in 175 cord blood samples in Japan. The seropositivity rate (≥1:32) for HPeV3 was 61%, similar to that for the other genotypes, but decreased significantly as maternal age increased (p<0.001). Furthermore, during the 2014 HPeV3 epidemic, prospective measurement of NATs to HPeV3 in 45 patients with severe diseases caused by HPeV3 infection showed low NATs (≤1:16) at onset and persistently high NATs (≥1:512) until age 6 months. All intravenous immunoglobulin samples tested elicited high NATs to HPeV3. Our findings indicate that maternal antibodies to HPeV3 may help protect young infants from severe diseases related to HPeV3 and that antibody supplementation may benefit these patients.

Comparison of JEV neutralization assay using pseudotyped JEV with the conventional plaque-reduction neutralization test

We previously reported the development of a neutralization assay system for evaluating Japanese Encephalitis Virus (JEV) neutralizing antibody (NAb) using pseudotyped-JEV (JEV-PV). JEV-PV-based neutralization assay offers several advantages compared with the current standard plaque-reduction neutralization test (PRNT), including simplicity, safety, and speed. To evaluate the suitability of the JEV-PV assay as new replacement neutralization assay, we compared its repeatability, reproducibility, specificity, and correlated its results with those obtained using the PRNT. These analyses showed a close correlation between the results obtained with the JEV-PV assay and the PRNT, using the 50% plaque reduction method as a standard for measuring NAb titers to JEV. The validation results met all analytical acceptance criteria. These results suggest that the JEV-PV assay could serve as a safe and simple method for measuring NAb titer against JEV and could be used as an alternative approach for assaying the potency of JEV neutralization.

Humoral Immunity to AAV-6, 8, and 9 in Normal and Dystrophic Dogs

Adeno-associated virus (AAV)-6, 8, and 9 are promising gene-delivery vectors for testing novel Duchenne muscular dystrophy gene therapy in the canine model. Humoral immunity greatly influences in vivo AAV transduction. However, neutralizing antibodies to AAV-6, 8, and 9 have not been systemically examined in normal and dystrophic dogs. To gain information on the seroprevalence of antibodies to AAV-6, 8, and 9, we measured neutralizing antibody titers using an in vitro transduction inhibition assay. We examined 72 naive serum samples and 26 serum samples obtained from dogs that had received AAV gene transfer. Our data demonstrated that AAV-6 neutralizing antibody was the most prevalent antibody in dogs irrespective of age, gender, disease status (dystrophic or not), and prior parvovirus vaccination history. Surprisingly, high-level anti-AAV-6 antibody was detected at birth in newborn puppies. Further, a robust antibody response was induced in affected, but not normal newborn dogs following systemic AAV gene transfer. Taken together, our data have provided an important baseline on the seroprevalence of AAV-6, 8, and 9 neutralizing antibodies in normal and Duchenne muscular dystrophy dogs. These results will help guide translational AAV gene-therapy studies in dog models of muscular dystrophy.

Duration of Neutralizing Antibody Titer after Japanese Encephalitis Vaccination

In paired serum samples collected from 17 children, we measured neutralizing antibody (NTAb) titers after the second series of routine Japanese encephalitis (JE) vaccination in Japan to estimate the duration of NTAb titer when children did not receive the third series of routine vaccination by applying a random coefficient model. We also measured NTAb titers in adult serum samples to confirm the duration of NTAb titer estimated in the analysis of pediatric serum samples. In the absence of the third series of routine vaccination, 18% (3/17), 47% (8/17), 82% (14/17) and 100% (17/17) of children were estimated to become NTAb negative at 5, 10, 15, and 20 years after the second series of routine vaccination, respectively. Of 38 adults, 39.5% (15/38) became NTAb negative; the percentage was somewhat lower than that of antibody-negative children. The results suggested that JE vaccination schedule should be reevaluated in the future.

Development of an improved method for measuring neutralizing antibody to rotavirus

An improved assay was developed for measuring neutralizing antibody titers against rotaviruses. This procedure used the same initial steps as performed to determine antibody titers by the focus reduction neutralization (FRN) assay. However, instead of counting infected cells after staining with fluorescein, reductions in virus infectivity by neutralizing antibody were determined by quantitation of viral antigen production using an ELISA. A linear relationship was found between ELISA absorbance values and focus forming units for each of four prototype rotaviruses, representative of serotypes 1–4. Thus, the serum dilution that resulted in neutralization of 60% of infectious virus (i.e. the neutralizing antibody titer) could be readily determined from absorbance values. Titers found by this method were similar to and as reproducible as those found by the FRN assay. Because this method is less laborious and the results are obtained by objective rather than subjective methods, it represents an improvement over the FRN assay.