LNP-CpG ODN-adjuvanted varicella-zoster virus glycoprotein E induced comparable levels of immunity with Shingrix™ in VZV-primed mice.

Latent varicella-zoster virus (VZV) may be reactivated to cause herpes zoster, which affects one in three people during their lifetime. The currently available subunit vaccine Shingrix™ is superior to the attenuated vaccine Zostavax® in terms of both safety and efficacy, but the supply of its key adjuvant component QS21 is limited. With ionizable lipid nanoparticles (LNPs) that were recently approved by the FDA for COVID-19 mRNA vaccines as carriers, and oligodeoxynucleotides containing CpG motifs (CpG ODNs) approved by the FDA for a subunit hepatitis B vaccine as immunostimulators, we developed a LNP vaccine encapsulating VZV-glycoprotein E (gE) and CpG ODN, and compared its immunogenicity with Shingrix™ in C57BL/6J mice. The results showed that the LNP vaccine induced comparable levels of gE-specific IgG antibodies to Shingrix™ as determined by enzyme-linked immunosorbent assay (ELISA). Most importantly, the LNP vaccine induced comparable levels of cell-mediated immunity (CMI) that plays decisive roles in the efficacy of zoster vaccines to Shingrix™ in a VZV-primed mouse model that was adopted for preclinical studies of Shingrix™. Number of IL-2 and IFN-γ secreting splenocytes and proportion of T helper 1 (Th1) cytokine-expressing CD4+ T cells in LNP-CpG-adjuvanted VZV-gE vaccinated mice were similar to that of Shingrix™ boosted mice. All of the components in this LNP vaccine can be artificially and economically synthesized in large quantities, indicating the potential of LNP-CpG-adjuvanted VZV-gE as a more cost-effective zoster vaccine.

Ionizable Lipid Nanoparticles Enhanced the Synergistic Adjuvant Effect of CpG ODNs and QS21 in a Varicella Zoster Virus Glycoprotein E Subunit Vaccine.

Varicella zoster virus (VZV) causes two diseases: varicella upon primary infection and herpes zoster when latent viruses in the sensory ganglia reactivate. While varicella vaccines depend on humoral immunity to prevent VZV infection, cell-mediated immunity (CMI), which plays a therapeutic role in the control or elimination of reactivated VZV in infected cells, is decisive for zoster vaccine efficacy. As one of the most abundant glycoproteins of VZV, conserved glycoprotein E (gE) is essential for viral replication and transmission between ganglion cells, thus making it an ideal target subunit vaccine antigen; gE has been successfully used in the herpes zoster vaccine ShingrixTM on the market. In this report, we found that ionizable lipid nanoparticles (LNPs) approved by the Food and Drug Administration (FDA) as vectors for coronavirus disease 2019 (COVID-19) mRNA vaccines could enhance the synergistic adjuvant effect of CpG oligodeoxynucleotides (CpG ODNs) and QS21 on VZV-gE, affecting both humoral immunity and CMI. Vaccines made with these LNPs showed promise as varicella vaccines without a potential risk of herpes zoster, which identifies them as a novel type of herpes zoster vaccine similar to ShingrixTM. All of the components in this LNP-CpG-QS21 adjuvant system were proven to be safe after mass vaccination, and the high proportion of cholesterol contained in the LNPs was helpful for limiting the cytotoxicity induced by QS21, which may lead to the development of a novel herpes zoster subunit vaccine for clinical application.

Th2-Oriented Immune Serum After SARS-CoV-2 Vaccination Does Not Enhance Infection In Vitro.

The relatively lower protection rate of the alum-adjuvanted inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines reminds us of the antibody-dependent enhancement (ADE) phenomenon observed in preclinical studies during the development of vaccines for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1). In this study, using the S1 segment of the SARS-CoV-2 spike protein or inactivated whole SARS-CoV-2 virus as an antigen and aluminum as an adjuvant, the risk of ADE of infection with T helper 2 (Th2)-oriented immune serum from mice (N=6) and humans (N=5) was examined in immune cell lines, which show different expression patterns of Fc receptors. Neither the immune serum from alum-adjuvanted S1 subunit vaccines nor inactivated SARS-CoV-2 vaccination enhanced SARS-CoV-2 S pseudotyped virus infection in any of the tested cell lines in vitro. Because both of these Th2-oriented immune sera could block SARS-CoV-2 infection without ADE of infection, we speculate that the lower protection rate of the inactivated SARS-CoV-2 vaccine may be attributed to the lower neutralizing antibody titers induced or the pulmonary eosinophilic immunopathology accompanied by eosinophilic infiltration in the lungs upon virus exposure. Adjustment of the immunization schedule to elevate the neutralizing antibody levels and skew adjuvants toward Th1-oriented responses may be considered to increase the efficacies of both inactivated and spike protein-based subunit SARS-CoV-2 vaccines.

Comparison of immune responses induced by two or three doses of an alum-adjuvanted inactivated SARS-CoV-2 vaccine in mice.

Waning antibodies and rapidly emerging variants are challenges for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine development. Adjusting existing immunization schedules and further boosting strategies are under consideration. Here, the immune responses induced by an alum-adjuvanted inactivated SARS-CoV-2 vaccine in mice were compared among immunization schedules with two or three doses. For the two-dose schedule, a 0-28-day schedule induced 5-fold stronger spike-specific IgG responses than a 0-14-day schedule, with only a slight elevation of spike-specific cellular immunity 14 days after the last immunization. A third homologous boost 2 or 5 months after the second dose for the 0-28-day schedule slightly strengthened humoral responses (1.3-fold for the 0-1-3-month schedule, and 1.8-fold for the 0-1-6-month schedule) 14 days after the last immunization. Additionally, a third homologous boost (especially with the 0-1-3-month schedule) induced significantly stronger cell-mediated immunity than both two-dose immunization schedules for all indexes tested, with a response similar to that induced by a one-dose heterologous boost with BNT162b2 in clinical trials, according to cellular immunity analysis (1.5-fold). These T cell responses were Th2 oriented, with good CD4+ and CD8+ memory. These results may offer clues for applying a homologous boosting strategy for alum-adjuvanted inactivated SARS-CoV-2 vaccines.

Effects of Varicella-Zoster Virus Glycoprotein E Carboxyl-Terminal Mutation on mRNA Vaccine Efficacy.

Glycoprotein E (gE) is one of the most abundant glycoproteins in varicella-zoster virus and plays pivotal roles in virus replication and transmission between ganglia cells. Its extracellular domain has been successfully used as an antigen in subunit zoster vaccines. The intracellular C-terminal domain was reported to be decisive for gE trafficking between the endoplasmic reticulum, trans-Golgi network and endosomes and could influence virus spread and virus titers. Considering that the trafficking and distribution of mRNA vaccine-translated gE may be different from those of gE translated against the background of the viral genome (e.g., most gE in virus-infected cells exists as heterodimers with another glycoprotein, gI,), which may influence the immunogenicity of gE-based mRNA vaccines, we compared the humoral and cellular immunity induced by LNP-encapsulated mRNA sequences encoding the whole length of gE, the extracellular domain of gE and a C-terminal double mutant of gE (mutant Y569A with original motif AYRV, which targets gE to TGN, and mutants S593A, S595A, T596A and T598A with the original motif SSTT) that were reported to enhance virus spread and elevate virus titers. The results showed that while the humoral and cellular immunity induced by all of the mRNA vaccines was comparable to or better than that induced by the AS01B-adjuvanted subunit vaccines, the C-terminal double mutant of gE showed stable advantages in all of the indicators tested, including gE-specific IgG titers and T cell responses, and could be adopted as a candidate for both safer varicella vaccines and effective zoster vaccines.

Associations of Ambient Air Pollutants and Meteorological Factors With COVID-19 Transmission in 31 Chinese Provinces: A Time Series Study.

Evidence regarding the effects of environmental factors on COVID-19 transmission is mixed. We aimed to explore the associations of air pollutants and meteorological factors with COVID-19 confirmed cases during the outbreak period throughout China. The number of COVID-19 confirmed cases, air pollutant concentrations, and meteorological factors in China from January 25 to February 29, 2020, (36 days) were extracted from authoritative electronic databases. The associations were estimated for a single-day lag as well as moving averages lag using generalized additive mixed models. Region-specific analyses and meta-analysis were conducted in 5 selected regions from the north to south of China with diverse air pollution levels and weather conditions and sufficient sample size. Nonlinear concentration-response analyses were performed. An increase of each interquartile range in PM2.5, PM10, SO2, NO2, O3, and CO at lag4 corresponded to 1.40 (1.37-1.43), 1.35 (1.32-1.37), 1.01 (1.00-1.02), 1.08 (1.07-1.10), 1.28 (1.27-1.29), and 1.26 (1.24-1.28) ORs of daily new cases, respectively. For 1°C, 1%, and 1 m/s increase in temperature, relative humidity, and wind velocity, the ORs were 0.97 (0.97-0.98), 0.96 (0.96-0.97), and 0.94 (0.92-0.95), respectively. The estimates of PM2.5, PM10, NO2, and all meteorological factors remained significantly after meta-analysis for the five selected regions. The concentration-response relationships showed that higher concentrations of air pollutants and lower meteorological factors were associated with daily new cases increasing. Higher air pollutant concentrations and lower temperature, relative humidity and wind velocity may favor COVID-19 transmission. Controlling ambient air pollution, especially for PM2.5, PM10, NO2, may be an important component of reducing risk of COVID-19 infection. In addition, as winter months are arriving in China, the meteorological factors may play a negative role in prevention. Therefore, it is significant to implement the public health control measures persistently in case another possible pandemic.

Waning antibodies from inactivated SARS-CoV-2 vaccination offer protection against infection without antibody-enhanced immunopathology in rhesus macaque pneumonia models.

Inactivated coronaviruses, including severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), as potential vaccines have been reported to result in enhanced respiratory diseases (ERDs) in murine and nonhuman primate (NHP) pneumonia models after virus challenge, which poses great safety concerns of antibody-dependent enhancement (ADE) for the rapid wide application of inactivated SARS-CoV-2 vaccines in humans, especially when the neutralizing antibody levels induced by vaccination or initial infection quickly wane to nonneutralizing or subneutralizing levels over the time. With passive transfer of diluted postvaccination polyclonal antibodies to mimic the waning antibody responses after vaccination, we found that in the absence of cellular immunity, passive infusion of subneutralizing or nonneutralizing anti-SARS-CoV-2 antibodies could still provide some level of protection against infection upon challenge, and no low-level antibody-enhanced infection was observed. The anti-SARS-CoV-2 IgG-infused group and control group showed similar, mild to moderate pulmonary immunopathology during the acute phase of virus infection, and no evidence of vaccine-related pulmonary immunopathology enhancement was found. Typical immunopathology included elevated MCP-1, IL-8 and IL-33 in bronchoalveolar lavage fluid; alveolar epithelial hyperplasia; and exfoliated cells and mucus in bronchioles. Our results corresponded with the recent observations that no pulmonary immunology was detected in preclinical studies of inactivated SARS-CoV-2 vaccines in either murine or NHP pneumonia models or in large clinical trials and further supported the safety of inactivated SARS-CoV-2 vaccines.

An Established Th2-Oriented Response to an Alum-Adjuvanted SARS-CoV-2 Subunit Vaccine Is Not Reversible by Sequential Immunization with Nucleic Acid-Adjuvanted Th1-Oriented Subunit Vaccines.

A recently reported parallel preclinical study between a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine and an inactivated SARS-CoV-2 vaccine adjuvanted with alum showed pulmonary immunopathology typical of eosinophil accumulation in a mouse pneumonia model for the latter, which implied a potential role of cellular immunity in the difference in the protection rate between these two forms of vaccines. For those who have been vaccinated with alum-adjuvanted subunit or inactivated SARS-CoV-2 vaccines, whether the Th2 responses that have been established and the absence of induced cellular immunity could be changed is an open question. Using two heterologous boosts with Th1-oriented CpG ODN-adjuvanted S1-based SARS-CoV-2 subunit vaccines for mice that were primed with two doses of Th2-oriented alum-adjuvanted S1-based SARS-CoV-2 subunit vaccines, we demonstrated that established Th2 orientation could not be reversed to Th1 orientation and that no cellular immunity was induced, which should have been induced if the boosting vaccines were used as the prime vaccines. These results remind us that if widely administered alum-adjuvanted SARS-CoV-2 vaccines cannot overcome the challenge of coronavirus disease 2019 (COVID-19) and that if cellular immunity is important for the efficacy of SARS-CoV-2 vaccines in the future, the choice of more powerful heterologous boosting vaccine forms that can induce cellular immunity should be considered very carefully before application.

SARS-CoV-2 S1 is superior to the RBD as a COVID-19 subunit vaccine antigen.

Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into a global pandemic within a matter of months. While subunit vaccines are one of the prominent options for combating coronavirus disease 2019 (COVID-19), the immunogenicity of spike protein-based antigens remains unknown. When immunized in mice, the S1 domain induced much higher IgG and IgA antibody levels than the receptor-binding domain (RBD) and more efficiently neutralized SARS-CoV-2 when adjuvanted with alum. It is inferred that a large proportion of these neutralization epitopes are located in the S1 domain but outside the RBD and that some of these are spatial epitopes. This finding indicates that expression systems with posttranslational modification abilities are important to maintain the natural configurations of recombinant spike protein antigens and are critical for effective COVID-19 vaccines. Further, adjuvants prone to a Th1 response should be considered for S1-based subunit COVID-19 vaccines to reduce the potential risk of antibody-dependent enhancement of infection.

N-terminally truncated nucleocapsid protein of SARS-CoV-2 as a better serological marker than whole nucleocapsid protein in evaluating the immunogenicity of inactivated SARS-CoV-2.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.

Immunogenicity of Varicella-Zoster Virus Glycoprotein E Formulated with Lipid Nanoparticles and Nucleic Immunostimulators in Mice.

Theoretically, the subunit herpes zoster vaccine ShingrixTM could be used as a varicella vaccine that avoids the risk of developing shingles from vaccination, but bedside mixing strategies and the limited supply of the adjuvant component QS21 have made its application economically impracticable. With lipid nanoparticles (LNPs) that were approved by the FDA as vectors for severe acute respiratory syndrome coronavirus 2 vaccines, we designed a series of vaccines efficiently encapsulated with varicella-zoster virus glycoprotein E (VZV-gE) and nucleic acids including polyinosinic-polycytidylic acid (Poly I:C) and the natural phosphodiester CpG oligodeoxynucleotide (CpG ODN), which was approved by the FDA as an immunostimulator in a hepatitis B vaccine. Preclinical trial in mice showed that these LNP vaccines could induce VZV-gE IgG titers more than 16 times those induced by an alum adjuvant, and immunized serum could block in vitro infection completely at a dilution of 1:80, which indicated potential as a varicella vaccine. The magnitude of the cell-mediated immunity induced was generally more than 10 times that induced by the alum adjuvant, indicating potential as a zoster vaccine. These results showed that immunostimulatory nucleic acids together with LNPs have promise as safe and economical varicella and zoster vaccine candidates.