A bivalent Epstein-Barr virus vaccine induces neutralizing antibodies that block infection and confer immunity in humanized mice.

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several human cancers and, more recently, multiple sclerosis. Despite its prevalence and health impact, there are currently no vaccines or treatments. Four viral glycoproteins (gp), gp350 and gH/gL/gp42, mediate entry into the major sites of viral replication, B cells, and epithelial cells. Here, we designed a nanoparticle vaccine displaying these proteins and showed that it elicits potent neutralizing antibodies that protect against infection in vivo. We designed single-chain gH/gL and gH/gL/gp42 proteins that were each fused to bacterial ferritin to form a self-assembling nanoparticle. Structural analysis revealed that single-chain gH/gL and gH/gL/gp42 adopted a similar conformation to the wild-type proteins, and the protein spikes were observed by electron microscopy. Single-chain gH/gL or gH/gL/gp42 nanoparticle vaccines were constructed to ensure product homogeneity needed for clinical development. These vaccines elicited neutralizing antibodies in mice, ferrets, and nonhuman primates that inhibited EBV entry into both B cells and epithelial cells. When mixed with a previously reported gp350 nanoparticle vaccine, gp350D123, no immune competition was observed. To confirm its efficacy in vivo, humanized mice were challenged with EBV after passive transfer of IgG from mice vaccinated with control, gH/gL/gp42+gp350D123, or gH/gL+gp350D123 nanoparticles. Although all control animals were infected, only one mouse in each vaccine group that received immune IgG had detectable transient viremia. Furthermore, no EBV lymphomas were detected in immune animals. This bivalent EBV nanoparticle vaccine represents a promising candidate to prevent EBV infection and EBV-related malignancies in humans.

Broad neutralization of H1 and H3 viruses by adjuvanted influenza HA stem vaccines in nonhuman primates.

Seasonal influenza vaccines confer protection against specific viral strains but have restricted breadth that limits their protective efficacy. The H1 and H3 subtypes of influenza A virus cause most of the seasonal epidemics observed in humans and are the major drivers of influenza A virus-associated mortality. The consequences of pandemic spread of COVID-19 underscore the public health importance of prospective vaccine development. Here, we show that headless hemagglutinin (HA) stabilized-stem immunogens presented on ferritin nanoparticles elicit broadly neutralizing antibody (bnAb) responses to diverse H1 and H3 viruses in nonhuman primates (NHPs) when delivered with a squalene-based oil-in-water emulsion adjuvant, AF03. The neutralization potency and breadth of antibodies isolated from NHPs were comparable to human bnAbs and extended to mismatched heterosubtypic influenza viruses. Although NHPs lack the immunoglobulin germline VH1-69 residues associated with the most prevalent human stem-directed bnAbs, other gene families compensated to generate bnAbs. Isolation and structural analyses of vaccine-induced bnAbs revealed extensive interaction with the fusion peptide on the HA stem, which is essential for viral entry. Antibodies elicited by these headless HA stabilized-stem vaccines neutralized diverse H1 and H3 influenza viruses and shared a mode of recognition analogous to human bnAbs, suggesting that these vaccines have the potential to confer broadly protective immunity against diverse viruses responsible for seasonal and pandemic influenza infections in humans.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

Development of a Pan-H1 Influenza Vaccine.

The efficacy of current seasonal influenza vaccines varies greatly, depending on the match to circulating viruses. Although most vaccines elicit strain-specific responses, some present cross-reactive epitopes that elicit antibodies against diverse viruses and remain unchanged and effective for several years. To determine whether combinations of specific H1 hemagglutinin (HA) antigens stimulate immune responses that protect against diverse H1 influenza viruses, we evaluated the antibody responses elicited by HA-ferritin nanoparticles derived from six evolutionarily divergent H1 sequences and two computationally optimized broadly reactive antigen (COBRA) HA antigens. Humoral responses were assessed against a panel of 16 representative influenza virus strains from the past 80 years. HAs from the strains A/NewCaledonia/20/1999 (NC99), A/California/04/2009 (CA09), A/HongKong/117/1977 (HK77), COBRA X6, or P1 elicited neutralization against diverse strains, and a combination of three wild-type HA or two COBRA HA nanoparticles conferred significant additional breadth beyond that observed with any individual strain. Therefore, combinations of H1 HAs may constitute a pan-H1 influenza vaccine.IMPORTANCE Seasonal influenza vaccines elicit strain-specific immune responses designed to protect against circulating viruses. Because these vaccines often show limited efficacy, the search for a broadly protective seasonal vaccine remains a priority. Among different influenza virus subtypes, H1N1 has long been circulating in humans and has caused pandemic outbreaks. In order to assess the potential of a multivalent HA combination vaccine to improve the breadth of protection against divergent H1N1 viruses, HA-ferritin nanoparticles were made and evaluated in mice against a panel of historical and contemporary influenza virus strains. Trivalent combinations of H1 nanoparticles improved the breadth of immunity against divergent H1 influenza viruses.

Screening of primary gp120 immunogens to formulate the next generation polyvalent DNA prime-protein boost HIV-1 vaccines.

Our previous preclinical studies and a Phase I clinical trial DP6-001 have indicated that a polyvalent Env formulation was able to elicit broadly reactive antibody responses including low titer neutralizing antibody responses against viral isolates of subtypes A, B, C and AE. In the current report, a panel of 62 gp120 immunogens were screened in a rabbit model to identify gp120 immunogens that can elicit improved binding and neutralizing antibody responses and some of them can be included in the next polyvalent formulation. Only about 19% of gp120 immunogens in this panel were able to elicit neutralizing antibodies against greater than 50% of the viruses included in a high throughput PhenoSense neutralization assay when these immuongens were tested as a DNA prime followed by a fixed 5-valent gp120 protein vaccine boost. The new polyvalent formulation, using five gp120 immunogens selected from this subgroup, elicited improved quality of antibody responses in rabbits than the previous DP6-001 formulation. More significantly, this new polyvalent formulation elicited higher antibody responses against a panel of gp70V1/V2 antigens expressing V1/V2 sequences from diverse subtypes. Bioinformatics analysis supports the design of a 4-valent or 5-valent formulation using gp120 immunogens from this screening study to achieve a broad coverage against 16 HIV-1 subtypes.

Levels of N-linked glycosylation on the V1 loop of HIV-1 Env proteins and their relationship to the antigenicity of Env from primary viral isolates.

A good understanding about the structure and function of the envelope glycoprotein (Env) from primary human immunodeficiency virus-1 (HIV-1) isolates is important in facilitating the development of effective neutralizing antibody responses as a component of an effective HIV-1 vaccine. In the current study, the antigenicity of a panel of diverse HIV-1 primary Env from different clades of HIV-1 Group M was analyzed using rabbit sera produced by either 3- or 9-valent gp120 DNA vaccine formulations. Both the 3- and 9-valent gp120 DNA vaccine formulations elicited HIV-1 gp120-specific antibodies in immunized rabbits. However, we observed two levels of primary envelope antigenicity to the same set of rabbit immune sera and that the level of glycosylation, particularly in the V1 loop, may contribute to such diversity. Bioinformatics analysis on the distribution and average number of the N-linked glycosylation sites in all variable regions (V1-V5) was conducted. A linear plot demonstrated that the average number of potential N-glycosylation sites in the V1 and V4 loops correlates to the size of the loop. These data provide further evidence on the complexity of primary HIV-1 Env antigens and offers new insight into the mechanisms that HIV-1 uses to escape protective immune responses.

The ability of Hepatitis B surface antigen DNA vaccine to elicit cell-mediated immune responses, but not antibody responses, was affected by the deglysosylation of S antigen.

Hepatitis B Virus (HBV) infection remains a major worldwide infectious disease with serious long-term morbidity and mortality. The limited selections of drug treatment are not able to control the progress of disease in people with active and persistent HBV infection. Immunotherapy to control the degree of viral infection is one possible alternative solution to this challenge. HBV DNA vaccines, with their strong ability to induce cell-mediated immune responses, offer an attractive option. HBV surface protein is important in viral immunity. Re-establishing anti-S immunity in chronic HBV infected patients will bring significant benefit to the patients. Previous studies have shown that HBV S DNA vaccines are immunogenic in a number of animal studies. In the current study, we further investigated the effect of glycosylation to the expression and immunogenicity of S DNA vaccines. Our results demonstrate that deglycosylation at the two potential N-linked glycosylation sites in S protein resulted in a significant decrease of S-specific cell-mediated immune responses, but did not affect anti-S antibody responses. This finding provides important direction to the development of S DNA vaccines to elicit the optimal and balanced antibody and cell-mediated immune responses to treat people with HBV chronic infections.

Immunogenicity and protection efficacy of subunit-based smallpox vaccines using variola major antigens.

The viral strain responsible for smallpox infection is variola major (VARV). As a result of the successful eradication of smallpox with the vaccinia virus (VACV), the general population is no longer required to receive a smallpox vaccine, and will have no protection against smallpox. This lack of immunity is a concern due to the potential for use of smallpox as a biological weapon. Considerable progress has been made in the development of subunit-based smallpox vaccines resulting from the identification of VACV protective antigens. It also offers the possibility of using antigens from VARV to formulate the next generation subunit-based smallpox vaccines. Here, we show that codon-optimized DNA vaccines expressing three VARV antigens (A30, B7 and F8) and their recombinant protein counterparts elicited high-titer, cross-reactive, VACV neutralizing antibody responses in mice. Vaccinated mice were protected from intraperitoneal and intranasal challenges with VACV. These results suggest the feasibility of a subunit smallpox vaccine based on VARV antigen sequences to induce immunity against poxvirus infection.

Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen.

Recent studies have established the feasibility of subunit-based experimental vaccines to protect animals from lethal poxvirus infection. Individual outer membrane proteins from intracellular and extracellular virions of vaccinia virus, when delivered in the form of either DNA vaccines or recombinant protein vaccines produced from baculovirus-infected insect cells, were able to protect mice from the vaccinia virus challenge and rhesus macaques from the monkeypox virus challenge. The polyvalent formulations with various combinations of the four poxvirus antigens (A27, L1, B5 and A33) achieved better protection than the monovalent formulation using only one of these antigens. However, it is not clear whether any of the remaining outer membrane poxvirus proteins can further improve the efficacy of the current polyvalent formulations. In this study, we conducted detailed analysis on the immunogenicity of D8, a previously reported protective antigen from intracellular mature virions. Our results indicated that D8 induced strong protective antibody responses and was effective in improving the efficacy of previously reported polyvalent poxvirus vaccine formulations. Therefore, D8 is an excellent candidate antigen to be included in the final polyvalent subunit-based poxvirus vaccines.

Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E.

A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations.

Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype.

The immunogenicity of a poylvalent HIV-1 vaccine comprised of Env antigens from primary R5 isolates was evaluated in rhesus macaques. DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 from multiple HIV-1 isolates that were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization. These sera neutralized homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV encoding Env from HIV-1(Ba-L), whereas the virus load was reduced in the remaining animals compared to naïve controls. Hence priming with DNA encoding Env antigens from multiple HIV-1 clades followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of R5 tropic SHIV isolate.

Preclinical evaluation of cellular immune responses elicited by a polyvalent DNA prime/protein boost HIV-1 vaccine.

While DNA vaccines have been shown to prime cellular immune responses, levels are often low in nonhuman primates or humans. Hence, efforts have been directed toward boosting responses by combining DNA with different vaccination modalities. To this end, a polyvalent DNA prime/protein boost vaccine, consisting of codon optimized HIV-1 env (A, B, C, E) and gag (C) and homologous gp120 proteins in QS-21, was evaluated in rhesus macaques and BALB/c mice. Humoral and cellular responses, detected following DNA immunization, were increased following protein boost in macaques and mice. In dissecting cellular immune responses in mice, protein-enhanced responses were found to be mediated by CD4+ and CD8+ T cells with a Th1 cytokine bias. Our study reveals that, in addition to augmenting humoral responses, protein boosting of DNA-primed animals augments cellular immune responses mediated by CD8+ CTL, CD4+ T-helper cells and Th1 cytokines; thus, offering much promise in controlling HIV-1 in vaccinees.

Relative contributions of codon usage, promoter efficiency and leader sequence to the antigen expression and immunogenicity of HIV-1 Env DNA vaccine.

Optimized antigen expression is critical to the immunogenicity of DNA vaccines. A number of approaches have been proposed to enhance the antigen expression and/or immunogenicity of DNA vaccines, but their relative contributions have not been compared in a same antigen system. In the current study, optimization of codon usage, enhancement of viral promoter function and selection of secretary leader sequences were evaluated for their roles in improving the immunogenicity of a same model antigen, the HIV-1 envelope glycoprotein. Our data demonstrated that all these factors can work synergistically to improve the final antigen expression and immunogenicity of HIV-1 Env DNA vaccines, indicating they work through different mechanisms. The best result came from the approach that optimized all three components in a DNA vaccine design. Our study further revealed that the levels of HIV-1 env-specific RNA transcripts in transiently transfected 293T cells were higher from the codon-optimized gene than the wild type counterpart. This finding suggested other mechanism may also contribute to the increased antigen expression and immunogenicity of codon-optimized DNA vaccines in addition to the improved tRNA usage in mammalian cells for codon-optimized viral genes as previously reported.

Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate.

Immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naïve animals. Further, one of six immunized animals was completely protected whereas all six naïve animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L).

Novel DNA vaccine based on hepatitis B virus core gene induces specific immune responses in Balb/c mice.

AIM: To investigate the immunogenicity of a novel DNA vaccine, pSW3891/HBc, based on HBV core gene in Balb/c mice. METHODS: A novel DNA vaccine, pSW3891/HBc, encoding HBV core gene was constructed using a vector plasmid pSW3891. Balb/c mice were immunized with either pSW3891/HBc or empty vector DNA via gene gun. IgG anti-HBc responses in mouse sera were demonstrated by ELISA. Specific cytotoxicity of cytotoxic T lymphocytes (CTLs) of mice was quantitatively measured by lactate dehydrogenase release assay. RESULTS: HBcAg was expressed effectively in 293T cell line transiently transfected with pSW3891/HBc. Strong IgG anti-HBc responses were elicited in mice immunized with pSW3891/HBc. The end-point titers of anti-HBc reached the highest 1:97,200, 4 wk after the third immunization. The specific CTL killing with the highest specific lysis reached 73.25% at effector:target ratio of 20:1 in mice that received pSW3891/HBc DNA vaccine. CONCLUSION: pSW3891/HBc vaccination elicits specific anti-HBc response and induces HBc-specific CTL response in immunized Balb/c mice.

Application of the polyvalent approach to HIV-1 vaccine development.

One major obstacle to the design of a global HIV-1 vaccine is viral diversity. Presently, data suggest that a single antigen will not suffice to generate broadly reactive neutralizing antibodies to protect all individuals against all subtypes of HIV-1 infection. While some of the neutralizing epitopes are identified in the constant regions of the HIV-1 envelope (Env) glycoprotein, many are localized to variable regions and differ conformationally from one virus to the next. The successes of polyvalent vaccine approaches against other antigenically variable pathogens encourage adoption of the same approach for HIV-1 vaccine design. The critical question is which envelope antigens should be combined in a vaccine cocktail to provide maximum protection against HIV-1. A review of the existing human vaccines based on the polyvalent principle is included here to provide a historical perspective for the current effort of developing a polyvalent HIV-1 vaccine. Data generated from several groups actively working on candidate polyvalent HIV-1 vaccines are summarized. Information presented in this review highlights the potential and importance of the polyvalent vaccine approach for the future development of an effective HIV-1 vaccine.

Assays for the assessment of neutralizing antibody activities against Severe Acute Respiratory Syndrome (SARS) associated coronavirus (SCV).

Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome (SARS) or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus (SCV). However, the current assay based on the cytopathic effect (CPE) which has been frequently cited in literature has several limitations. The CPE assay relies on the visual observation on the damage of SCV infected target cells under a microscope. It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE. In the current study, we established the utility of two additional assays to measure the neutralizing activities against SCV: the plaque reduction (PR) and the neutral red staining (NRS) assays. The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells. The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). In this assay, the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A540. The neutralizing antibody titers can be easily determined with either of the two new assays. In this report, we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV.

Enhanced immunogenicity of gp120 protein when combined with recombinant DNA priming to generate antibodies that neutralize the JR-FL primary isolate of human immunodeficiency virus type 1.

Strategies are needed for human immunodeficiency virus type 1 vaccine development that improves the neutralizing antibody response against primary isolates of the virus. Here we examined recombinant DNA priming followed by subunit protein boosting as a strategy to generate neutralizing antibodies. Both plasmid-based and recombinant protein envelope (Env) glycoprotein immunogens were derived from a primary viral isolate, JR-FL. Serum from rabbits immunized with either gp120 or gp140 DNA vaccines delivered by gene gun inoculation followed by recombinant gp120 protein boosting was capable of neutralizing JR-FL. Neither the DNA vaccines alone nor the gp120 protein alone generated a detectable neutralizing antibody response against this virus. Neutralizing antibody responses using gp120 DNA and gp140 DNA for priming were similar. The results suggest that Env DNA priming followed by gp120 protein boosting provides an advantage over either approach alone for generating a detectable neutralizing antibody response against primary isolates that are not easily neutralized.

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV).

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.

Identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system.

The Spike (S) protein of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) plays important roles in viral pathogenesis and potentially in the development of an effective vaccine against this virulent infectious disease. In this study, the codon-optimized S gene of SARS-CoV was synthesized to construct DNA vaccine plasmids expressing either the full-length or segments of the S protein. High titer S-specific immunoglobulin G antibody responses were elicited in rabbits immunized with DNA against various segments of the S protein. Two neutralizing domains were identified on the S protein, one at the N terminus (Ser12-Thr535) and the other near the C terminus (Arg797-Ile1192).