Goats with low levels of AAV antibody may serve as candidates for large animal gene therapy.

AAV vector-mediated gene therapy has been proposed as a feasible strategy for several eye diseases. However, AAV antibodies in the serum prior to treatment hinder the transduction efficiency and thus the therapeutic effect. Therefore, it is necessary to evaluate AAV antibodies in the serum before gene therapy. As large animals, goats are more closely related to humans than rodents and more economically available than nonhuman primates. Here, we first evaluated the AAV2 antibody serum level in rhesus monkeys before AAV injection. Then, we optimized a cell-based neutralizing antibody assay for detecting AAV antibodies in the serum of Saanen goats and evaluated the consistency of the cell-based neutralizing antibody assay and ELISA for goat serum antibody evaluation. The cell-based neutralizing antibody assay showed that the percentage of macaques with low antibody levels was 42.86%; however, there were no macaques with low antibody levels when the serum was evaluated by ELISA. The proportion of goats with low antibody levels was 56.67% according to the neutralizing antibody assay and 33. 33% according to the ELISA, and McNemar's test showed that the results of the two assays were not significantly different (P = 0.754), but that their consistency is poor (Kappa = 0.286, P = 0.114). Moreover, longitudinal evaluation of serum antibodies before and after intravitreal injection of AAV2 in goats revealed that the level of AAV antibodies increased and transduction inhibition subsequently increased, as reported in humans, indicating that transduction inhibition should be taken into account at different stages of gene therapy. In summary, starting with an evaluation of monkey serum antibodies, we optimized a detection method of goat serum antibodies, providing an alternative large animal model for gene therapy, and our serum antibody measurement method may be applied to other large animals.

Effects of Severe Acute Respiratory Syndrome Coronavirus 2 Strain Variation on Virus Neutralization Titers: Therapeutic Use of Convalescent Plasma.

We compared neutralizing antibody titers of convalescent samples collected before and after the emergence of novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), against the wild-type virus and Alpha (B.1.1.7) and Beta (B.1.351) variants. Plasma samples collected in 2020 before emergence of variants showed reduced titers against the Alpha variants, and both sets of samples demonstrated significantly reduced titers against Beta. Comparison of microneutralization titers with those obtained with pseudotype and hemagglutination tests showed a good correlation between their titers and effects of strain variation, supporting the use of these simpler assays for assessing the potency of convalescent plasma against currently circulating and emerging strains of SARS-CoV-2.

Humoral Immunogenicity of the mRNA-1273 Vaccine in the Phase 3 Coronavirus Efficacy (COVE) Trial.

BACKGROUND: Messenger RNA (mRNA)-1273 vaccine demonstrated 93.2% efficacy against coronavirus disease 2019 (COVID-19) in the Coronavirus Efficacy (COVE) trial. The humoral immunogenicity results are now reported. METHODS: Participants received 2 mRNA-1273 (100 µg) or placebo injections, 28 days apart. Immune responses were evaluated in a prespecified, randomly selected per-protocol immunogenicity population (n = 272 placebo; n = 1185 mRNA-1273). Serum binding antibodies (bAbs) and neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-spike protein were assessed at days 1, 29, and 57 by baseline SARS-CoV-2-negative (n = 1197) and SARS-CoV-2-positive (n = 260) status, age, and sex. RESULTS: SARS-CoV-2-negative vaccinees had bAb geometric mean AU/mL levels of 35 753 at day 29 that increased to 316 448 at day 57 and nAb inhibitory dilution 50% titers of 55 at day 29 that rose to 1081 at day 57. In SARS-CoV-2-positive vacinees, the first mRNA-1273 injection elicited bAb and nAb levels that were 11-fold (410 049) and 27-fold (1479) higher than in SARS-CoV-2-negative vaccinees, respectively, and were comparable to levels after 2 injections in uninfected participants. Findings were generally consistent by age and sex. CONCLUSIONS: mRNA-1273 elicited robust serologic immune responses across age, sex, and SARS-CoV-2 status, consistent with its high COVID-19 efficacy. Higher immune responses in those previously infected support a booster-type effect. Clinical Trials Registration. NCT04470427.

Stochastic Interventional Vaccine Efficacy and Principal Surrogate Analyses of Antibody Markers as Correlates of Protection against Symptomatic COVID-19 in the COVE mRNA-1273 Trial.

The COVE trial randomized participants to receive two doses of mRNA-1273 vaccine or placebo on Days 1 and 29 (D1, D29). Anti-SARS-CoV-2 Spike IgG binding antibodies (bAbs), anti-receptor binding domain IgG bAbs, 50% inhibitory dilution neutralizing antibody (nAb) titers, and 80% inhibitory dilution nAb titers were measured at D29 and D57. We assessed these markers as correlates of protection (CoPs) against COVID-19 using stochastic interventional vaccine efficacy (SVE) analysis and principal surrogate (PS) analysis, frameworks not used in our previous COVE immune correlates analyses. By SVE analysis, hypothetical shifts of the D57 Spike IgG distribution from a geometric mean concentration (GMC) of 2737 binding antibody units (BAU)/mL (estimated vaccine efficacy (VE): 92.9% (95% CI: 91.7%, 93.9%)) to 274 BAU/mL or to 27,368 BAU/mL resulted in an overall estimated VE of 84.2% (79.0%, 88.1%) and 97.6% (97.4%, 97.7%), respectively. By binary marker PS analysis of Low and High subgroups (cut-point: 2094 BAU/mL), the ignorance interval (IGI) and estimated uncertainty interval (EUI) for VE were [85%, 90%] and (78%, 93%) for Low compared to [95%, 96%] and (92%, 97%) for High. By continuous marker PS analysis, the IGI and 95% EUI for VE at the 2.5th percentile (519.4 BAU/mL) vs. at the 97.5th percentile (9262.9 BAU/mL) of D57 Spike IgG concentration were [92.6%, 93.4%] and (89.2%, 95.7%) vs. [94.3%, 94.6%] and (89.7%, 97.0%). Results were similar for other D29 and D57 markers. Thus, the SVE and PS analyses additionally support all four markers at both time points as CoPs.

A comparative multi-tiered immunogenicity assessment of biosimilar pegylated filgrastim: validation of methods for clinical assessment of INTP5.

BACKGROUND AND OBJECTIVE: Validated and highly sensitive assays are required for comparative assessment of immunogenicity of biosimilars. For INTP5, a biosimilar pegylated filgrastim, the immunogenicity assessment included tiers that allowed for assessment of antibodies against the PEG and the Filgrastim moieties for comparative clinical immunogenicity assessment. METHODS: Electrochemiluminescence immunoassay (ECLIA) was used for Screening, Specificity, and Titer assays for detecting anti-drug antibodies (ADAs) and cell-based method for neutralizing ADAs. The methods were validated to enable use of same methods irrespective of biosimilar or reference arms. RESULTS: The ADA and cell-based assay for neutralizing antibody detection were validated with a sensitivity capable of detecting binding Anti-Pegfilgrastim antibody at ~40 ng/mL and Neutralizing antibody at ~380 ng/mL and used for a comparative immunogenicity study. Of 194 subjects, 10 subjects had confirmed positive anti-drug-antibody in the biosimilar arm and 9 in the reference arm. None of the subjects were detected with neutralizing anti-drug antibodies. CONCLUSION: This work demonstrates the application of a rigorous approach toward validation of assays for immunogenicity studies for biosimilars. Highly sensitive, precise, and robust assays were used to conclude comparable low incidences of anti-drug antibodies in both biosimilar and innovator arms of the clinical study for Pegfilgrastim.

Cell-Based Determination of Neutralizing Antibodies Against Adeno-Associated Virus in Cardiac Gene Therapy.

The field of cardiac gene therapy has seen the rising use of adeno-associated viral (AAV) vectors as a promising therapeutic option for cardiac diseases and heart failure. To achieve intended results of AAV delivery, a majority of clinical studies screen patients for existing neutralizing antibodies that could inhibit the effects of the administered AAV and confound treatment efficacy. The cell-based neutralizing antibody assay offers a method of quantifying and identifying a patient's existing neutralizing antibodies against specific serotypes. Combined with the luciferase assay, the neutralizing antibody assay tests the ability of patient antibodies in the blood to prevent gene transduction of AAV-encoded luciferase gene at ranging serial dilutions. This chapter provides a protocol and experimental techniques to determine the presence of neutralizing antibodies against AAV in the blood.

MSD-based assays facilitate a rapid and quantitative serostatus profiling for the presence of anti-AAV antibodies.

Adeno-associated virus (AAV) vector applications are often limited by capsid-directed humoral immune responses, mainly through neutralizing antibodies (NAbs), which are present throughout the human population due to natural AAV infections. Currently, antibody levels are often quantified via ELISA-based protocols or by cellular NAb assays and less frequently by in vivo NAb assays in mice. These methods need optimization for each serotype and are often not applicable to AAV variants with poor in vitro transduction. To tackle these limitations, we have established Meso Scale Discovery (MSD)-based assays for the quantification of binding antibodies (BAbs) and NAbs against the three most commonly used AAV serotypes, AAV2, AAV8, and AAV9. Both assays detect anti-AAV-IgG1-3 with high sensitivity and consistency as shown in a screen of sera from 40 healthy human donors. Subsequently, BAb and NAb titers were determined for identification of seronegative animals in a non-human primate (NHP) cohort. Moreover, the MSD-based BAb assay protocol was extended to a panel of 14 different AAV serotypes. In summary, our platform allows a rapid and quantitative assessment of the immunological properties of any natural or engineered AAV variant irrespective of transduction efficiency and enables high-throughput screens.

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay.

Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

Neutralizing Antibodies Against Adeno-Associated Virus (AAV): Measurement and Influence on Retinal Gene Delivery.

Adeno-associated viral vectors have become widely used in the clinic for retinal gene therapy. Thanks to AAVs impeccable safety profile and positive functional outcomes in its clinical application, interest in retinal gene therapy has increased exponentially over the past decade. Although early clinical trials have shown there is little influence of neutralizing antibodies on the performance of AAV when vector is administered into the subretinal space, recent findings suggest neutralizing antibodies may play a role when AAV is delivered via the intravitreal route. These findings highlight the importance of microenvironment on gene delivery and stress the need for a versatile assay to screen subjects for the presence of AAV-neutralizing antibodies. Measuring NAb titers against AAV prior and after gene therapy will help us better understand the impact of preexisting immunity on gene transfer, especially when the vector is administered intravitreally.