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. 2023 Sep 22;11(5):e0159823.
doi: 10.1128/spectrum.01598-23. Online ahead of print.

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization

Affiliations

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization

Tal Noy-Porat et al. Microbiol Spectr. .

Abstract

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

Keywords: Mpox; VIG; neutralizing antibodies; orthopoxviruses; vaccinia virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Immunization and serum characterization. Two NHP females were immunized with live VACV. Animal 8126 (A) received six doses of Lister strain (gray arrows), while animal 3026 (B) received five doses of Lister (gray arrows) and additional three boosts of WR strain (red arrows). The numbers on the bottom of each arrow indicate the time of each boost (in weeks from the beginning of immunization). Binding titer (Dil50) and neutralization titer (NT50) were measured 7 d after each boost, using ELISA against VACV IHD-J or neutralization assay against VACV Lister, accordingly. (C and D) Serum samples of animals 8126 (C) and 3026 (D), collected at the end point of immunization, were evaluated for their ability to neutralize different VACV strains, as indicated in the figure. A commercial VIG sample, tested on the Lister strain, was used as a control. (E) The same serum samples were tested for their ability to neutralize two Mpox strains, 2018 and 2022, using an in vitro MV neutralization test. (F) Summary of NT50 values resulting from neutralization tests described in (C–E). The significance between the neutralization curves of animals 8126 vs 3026 was tested using a one-way ANOVA on the AUC and Tukey’s post test. ns, not significant; **, P < 0.003; ^, significance compared to other strains in the same animal (P < 0.05). All data points represent the mean ± SEM of triplicates.
FIG 2
FIG 2
Antibody profile analysis of VACV-immunized NHPs’ sera. Serum samples taken from two animals, 8126 and 3026, before the onset of immunization and 21 and 48 wk after the onset of immunization (t = 0, t = 21, and t = 48, respectively), were analyzed against an array of 224 vaccinia proteins. Commercial VIG was used for comparison. Results obtained for the 50 strongest proteins are shown as a heat map according to the scale in the figure (numbers represent fold change vs naives). In bold are the five central membrane proteins known to elicit a protective immune response. Statistical analysis was performed between the different time points for both NHPs (0–21, 21–48 wk, and VIG compared to 48 wk) using a paired t-test. ns, not significant; **, P < 0.003.
FIG 3
FIG 3
In vitro neutralization of monoclonal antibodies. Antibodies selected from the phage-display library were subjected to an in vitro MV neutralization assay using VACV-WR-vFIRE. The results of the most potent mAbs are presented in (A and B). All data points represent the mean ± SEM of triplicates. NT50 values obtained from this assay are summarized in (C). Antibodies that failed to exhibit MV neutralization were tested in an EV comet-inhibition assay. Antibodies presenting neutralization in this assay are presented in (D). Each mAb, indicated to the left of the wells, was tested at four concentrations (2.5–20 µg/mL), and the two lowest concentrations, exhibiting effective comet-inhibition, are presented (indicated at the top). A VIG sample was used as a reference in each MV and EV test, and a virus-only sample was used as a negative control. The assay was repeated twice for each mAb.
FIG 4
FIG 4
Binding characteristics of selected antibodies. (A) The specificity of the selected antibodies was determined by ELISA against the indicated proteins and against VACV IHD-J as a control. A VIG sample was used as a positive control. Data represent the mean ± SEM of triplicates. Results above the threshold of 0.1 O.D. (represented by a red line) were considered positive. (B) The binding profile of antibodies was determined by ELISA using VACV IHD-J as the coating antigen. Data represent the average of triplicates ± SEM. (C) Binding characteristics of the antibodies were determined using BLI. All antigens carrying a His tag were immobilized to Ni-NTA sensors and interacted with increasing amounts of the relevant antibody. Binding kinetics were fitted using a 1:1 binding model. (D and E) Epitope binning experiments were conducted using BLI. The antigen was immobilized on the Ni-NTA sensor and saturated with the first antibody. The complex was then incubated with each of the indicated antibodies. Time 0 represents the binding to the mAb1-antigen complex. Binding was evaluated by the ability of each pair of antibodies to simultaneously bind the antigen. (D) Binding of mAbs to the D8 protein. Each pair of antibodies, indicated in the legend, was tested separately and is indicated according to the order of binding. A curve showing each individual mAb binding to D8 without competition is shown for comparison. (E) Binding of mAbs to H3 protein. Each of the indicated mAbs was allowed to bind first, and the graph represents the binding of MV32 to the mAb1-H3 complex.
FIG 5
FIG 5
Mpox in vitro neutralization. Representative mAbs, MV32 and MV33, were tested for their ability to neutralize Mpox strains 2018 (A) and 2022 (B) in an in vitro MV neutralization assay. VIG samples were used as controls. Data represent the mean ± SEM of duplicates.

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