key: cord-0426767-583yl9tl
authors: Seo, Yong Bok; Shin, Duckhyang; Suh, You Suk; Na, Juyoung; Ryu, Ji In; Sung, Young Chul
title: Marked enhancement of neutralizing antibody and IFN-γ T-cell responses by GX-19N DNA booster in mice primed with inactivated vaccine
date: 2021-11-04
journal: bioRxiv
DOI: 10.1101/2021.11.02.467026
sha: 02837054db885371c66dff066f0a896b04d6c427
doc_id: 426767
cord_uid: 583yl9tl

In response to the COVID-19 pandemic, an unprecedented level of vaccine development has occurred. As a result, various COVID-19 vaccines have been approved for use. Among these, inactivated virus particle (VP) vaccines have been widely used worldwide, but additional vaccination strategies are needed because of the short duration of immune responses elicited by these vaccines. Here, we evaluated homologous and heterologous prime–boost regimens using a VP vaccine and GX-19N DNA vaccine for their ability to enhance the protective immune response against SARS-CoV-2. We demonstrated that a heterologous prime–boost regimen with the VP vaccine and GX-19N DNA vaccine resulted in enhanced SRBD- & N-specific antibody responses, compared to the homologous VP vaccine prime–boost vaccination. In addition, the neutralizing antibody response was significantly improved with the heterologous VP prime–DNA boost regimen, and the neutralizing antibody induced with the heterologous prime–boost regimen did not decrease against the SARS-CoV-2 variant of concern (VOC). The heterologous VP prime–DNA boost regimen not only significantly increased S- and N-specific IFN-γ T-cell responses, but also induced an equivalent level of T-cell response against SARS-CoV-2 VOCs. Our results provide new insights into prophylactic vaccination strategies for COVID-19 vaccination.

There has been an unprecedented level of vaccine development in response to the COVID- 19 pandemic. As a result, 22 COVID-19 vaccines have been approved for use, and more than 3.7 billion doses have been administered. This rapid development of an effective vaccine against COVID- 19 and its distribution to the general population has proven to be a very successful strategy for reducing the transmission and disease burden of COVID-19.

Among several vaccine platforms, inactivated virus particle (VP) vaccines have been extensively studied. They are generally safe and are widely used to prevent respiratory infections such as influenza and other infectious diseases such as hepatitis A, polio, and rabies 1 . VP vaccines are manufactured through an inactivation process of the virus, which leads to a loss of infectivity but retains the main viral antigenicity. VP vaccines also have the advantage of being easily stored and transported for years at 2-8 °C, making them suitable for use in many low-income countries and locations with limited cold storage capacity. However, production rates may be limited by the yield of the virus in cell culture and the requirements for a production facility at biosafety level 3.

The COVID-19 VP vaccines BBIBP-CorV (Sinopharm Beijing), Coronavac (Sinovac), and BBV152

(Bharat Biotech) have demonstrated protective efficacies of 78.1% 2 , 50.7% 3 , and 77.8% 4 , respectively, and have been approved for emergency use in several countries. As a result, a significant portion of the world's population has received a two-dose emergency immunization with one of the COVID-

The DNA vaccine is an effective platform for rapid control of the SARS-CoV-2 outbreak as it can be completed in weeks to produce a clinical-grade vaccine 5 . In addition, DNA vaccines have the advantage of being highly stable at various temperatures, making them suitable for delivery in environments where resources are limited and there are no cold chains 6 . It has been reported that unlike conventional vaccines, DNA vaccines can effectively stimulate both cellular and humoral responses to pathogens in a challenge model 7 . In addition, the potential of prophylactic DNA vaccines against viral infections such as MERS-CoV and ZIKV has been demonstrated in recent clinical trials 8, 9, 10, 11 . As the COVID-19 pandemic has spread worldwide, recent studies have reported that DNA vaccines induce antigen-specific T-cell responses, neutralize antibodies, and further protect animals from SARS-CoV-2 challenge 12, 13, 14 . A COVID-19 DNA vaccine, ZyCoV-D, has recently been approved for emergency use as it has shown 66.6% protective efficacy in phase 3 clinical trials 15 . In addition, two, eight, and one candidate DNA vaccines are currently in phases 3, 2, and 1 of clinical trials, respectively 16 .

A heterologous prime-boost has been shown to effectively improve the immunogenicity of HIV-1 and influenza vaccines 17, 18, 19, 20 . Heterologous prime-boost regimens using DNA and VP In this study, six-week-old female mice were primed with a VP vaccine and boosted with the VP vaccine or GX-19N DNA vaccine four weeks later for booster vaccination. Immune responses were evaluated two weeks after the last vaccination. The Spike-specific binding antibody was evaluated by ELISA using the receptor binding domain (S RBD , Wuhan) and nucleocapsid (N, Wuhan) proteins. The heterologous VP vaccine prime-GX-19N DNA vaccine boost induced a significantly higher S RBD -specific antibody response than the homologous VP vaccine prime-boost regimen. Compared to VP prime, VP boost increased antibody titer by 1.7fold, whereas DNA boost increased antibody titer by 181-fold. (Fig. 1A) . The ratio of IgG2a to IgG1 antibody titer was, albeit statistically insignificant, increased after GX-19N boosting (Fig 1B) , indicating the enhancement of Th1-polarized immunity, as reported previously 22, 23 . Likewise, the heterologous prime-boost vaccination also induced significantly higher NP-specific antibody responses than the homologous prime-boost vaccination. VP-prime followed by VP-boost increased antibody titer by 1.4-fold, whereas GX-19N DNA-boost increased antibody titer by 512fold. (Fig. 1C ). In addition, we evaluated the neutralizing antibody responses to the Wuhan and VOCs (B.1.351 and B.1.617.2) using the surrogate virus neutralization test (sVNT), which is highly correlated with the conventional virus neutralization test (cVNT) and pseudovirus-based VNT (pVNT) 24 . In contrast to the previous report that the boosting with 2 g VP vaccine increased about 2-fold neutralization titer, the booster immunization with VP vaccine in our experiment did not further enhance neutralization antibody responses in mice primed with VP vaccine. This discrepancy may be due to the differences in immunization dose (0.4 g vs. 2 g), and assay methods for evaluating neutralization antibody responses (sVNT vs. cVNT).

Heterologous vaccination induced a significantly higher neutralizing antibody titer than the homologous VP prime-boost vaccination. The neutralizing antibody titer obtained through the T-cell responses were also evaluated following the homologous and heterologous vaccination regimens. A higher Wuhan SARS-CoV-2 spike-specific IFN-γ response was detected by ELIspot in mice that received a heterologous vaccination regimen. Compared to VP-prime, VP-boost increased T cell response by 1.6-fold, whereas GX-19N DNAboost increased T cell response by 38.3fold. (Fig. 3A) . As expected, we observed similar levels of cellular responses to the B.1.351 (mean = 1,644 SFUs/10 6 splenocytes) and B.1.617.2 (mean = 1,753 SFUs/10 6 splenocytes) spike peptides (Fig.   3B ). Similar to the results for the spike peptides, a higher N-specific IFN-γ response was observed in the heterologous VP vaccine prime-GX-19N DNA vaccine boost regimen. VP-prime followed by VP-boost increased cellular response by 2.9-fold, whereas GX-19N DNA-boost increased cellular response by 8.8-fold. (Fig. 4A) . We also observed similar levels of IFN-γ T cell response to B.1.351 (mean = 739 SFUs/10 6 splenocytes) and B.1.617.2 (mean = 794 SFUs/10 6 splenocytes) nucleocapsid peptides (Fig. 4B) . This is consistent with previous findings that cellular immunity is relatively unimpaired by VOCs compared to neutralizing antibody responses 26 Comparison of data between groups was performed using two-tailed Student's t-tests. Statistical significance was set at P <0.05. Balb/c mice (n=4/group) were immunized at weeks 0 and 4 as described in the methods, and antibody responses were measured in the sera of mice collected 2 weeks after the last immunization.

Graphs show SARS-CoV-2 S RBD -specific IgG titer (A), ratios of S RBD -specific IgG2a to IgG1 titer (B) and SARS-CoV-2 N-specific IgG titer (C). Individual mice were represented by a single data point.

P-values were determined by two-tailed Student's t-test. *p<0.05. Balb/c mice (n=4/group) were immunized at weeks 0 and 4 as described in the methods, and antibody responses were measured in the sera of mice collected 2 weeks after the last immunization.

Sera from vaccinated mice were tested for sVNT 20 

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