Neutralizing Antibodies In Guinea Pigs Research Articles

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

A novel MHC-II targeted BVDV subunit vaccine induces a neutralizing immunological response in guinea pigs and cattle.

Bovine viral diarrhoea virus (BVDV) is a major cause of economic loss in the cattle industry, worldwide. Infection results in reduced productive performance, growth retardation, reduced milk production and increased susceptibility to other diseases leading to early culling of animals. There are two primary methods used to control the spread of BVDV: the elimination of persistently infected (PI) animals and vaccination. Currently, modified live or inactivated vaccines are used in BVDV vaccination programmes, but there are safety risks or insufficient protection, respectively, with these vaccines. Here, we report the development and efficacy of the first targeted subunit vaccine against BVDV. The core of the vaccine is the fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the major histocompatibility type II molecule (MHC-II) present on antigen-presenting cells. Industrial production of the vaccine is carried out using the baculovirus expression vector system (BEVS) using single-use manufacturing technologies. This new subunit vaccine induces strong BVDV-specific neutralizing antibodies in guinea pigs and cattle. Importantly, in cattle with low levels of natural BVDV-specific neutralizing antibodies, the vaccine induced strong neutralizing antibody levels to above the protective threshold, as determined by a competition ELISA. The APCH-E2 vaccine induced a rapid and sustained neutralizing antibody response compared with a conventional vaccine in cattle.

Immunogenicity and protective efficacy of a novel foot-and-mouth disease virus empty-capsid-like particle with improved acid stability

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects cloven-hoofed animal species. The FMDV capsid is highly acid labile and viral particles lose their immunogenicity when they disassemble at mildly acidic pHs. The viral capsid of FMDV serotype O is more sensitive than those of other serotypes, making it more difficult to acquire enough empty-capsid-like particles in the acidic insect cell environment for research. In this study, novel FMDV mutants with increased acid resistance were isolated using BHK-21 cell cultured under low-pH conditions. Amino acid substitutions Q25R, K41E, and N85A in the VP1 capsid protein and K154Q in the VP3 capsid protein were detected in all six mutants. Based on these amino acid replacements, empty-capsid-like particles of FMDV serotype O, which were resistant to the acid-induced dissociation of the capsid into pentameric subunits, were produced in insect cells. We characterized the protective immunity induced by these acid-resistant empty capsid particles. Significant humoral and cellular immune responses were elicited in mice after immunization with the acid-resistant empty capsid particles. The acid-resistant empty-capsid-like particles also induced strong neutralizing antibodies in guinea pigs and protected all the guinea pigs from FMDV challenge. Our results suggest that these acid-resistant empty-capsid-like particles have potential utility as a vaccine against serotype O FMDV infection.

Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses

African horse sickness (AHS) is caused by multiple serotypes of the dsRNA AHSV and is a major scourge of domestic equids in Africa. While there are well established commercial live attenuated vaccines produced in South Africa, risks associated with these have encouraged attempts to develop new and safer recombinant vaccines. Previously, we reported on the immunogenicity of a plant-produced AHS serotype 5 virus-like particle (VLP) vaccine, which stimulated high titres of AHS serotype 5-specific neutralizing antibodies in guinea pigs. Here, we report a similar response to the vaccine in horses. This is the first report demonstrating the safety and immunogenicity of plant-produced AHS VLPs in horses.

An experimental subunit vaccine based on Bluetongue virus 4 VP2 protein fused to an antigen-presenting cells single chain antibody elicits cellular and humoral immune responses in cattle, guinea pigs and IFNAR(−/−) mice

Bluetongue virus (BTV), the causative agent of bluetongue disease (BT) in domestic and wild ruminants, is worldwide distributed. A total of 27 serotypes have been described so far, and several outbreaks have been reported. Vaccination is critical for controlling the spread of BTV. In the last years, subunit vaccines, viral vector vaccines and reverse genetic-based vaccines have emerged as new alternatives to conventional ones. In this study, we developed an experimental subunit vaccine against BTV4, with the benefit of targeting the recombinant protein to antigen-presenting cells. The VP2 protein from an Argentine BTV4 isolate was expressed alone or fused to the antigen presenting cell homing (APCH) molecule, in the baculovirus insect cell expression system. The immunogenicity of both proteins was evaluated in guinea pigs and cattle. Titers of specific neutralizing antibodies in guinea pigs and cattle immunized with VP2 or APCH-VP2 were high and similar to those induced by a conventional inactivated vaccine. The immunogenicity of recombinant proteins was further studied in the IFNAR(−/−) mouse model where the fusion of VP2 to APCH enhanced the cellular immune response and the neutralizing activity induced by VP2.

HIV-1 envelope trimer elicits higher neutralizing antibody responses than monomeric gp120

Background HIV-1 envelope glycoprotein is the primary target for HIV1-specific antibodies. The native HIV-1 envelope spike on the virion surface is a trimer, but trimeric gp140 and monomeric gp120 are currently believed to induce comparable immune responses. Indeed, most studies on the immunogenicity of HIV-1 envelope oligomers have revealed only marginal improvement over monomers. We report here that stable and homogenous envelope trimers with characteristics expected for the native viral spikes are substantially superior at eliciting neutralizing antibodies in guinea pigs. Methods Stable envelope gp140 trimer derived from clinical isolate sequences were stabilized with the T4-fibritin C-terminal trimerization tag and produced in stablely transfected 293T cells. Characterization of Env trimers was performed by Western blotting, size-exclusion chromatography (SEC), analytical-ultra centrifugation (AUC), multi-angle light scattering (MALS) and surface plasmon resonance (SPR). Guinea pigs were immunized six times with 100 µg of protein trimer or monomer in CpG/Emulsigen adjuvants. Antibody responses were determined by ELISA and TZM.bl neutralizing antibody assays. Results Homogeneous trimer and monomer preparations exhibited high purity as measured by SEC and SDS-PAGE. AUC and MALS analyses revealed expected molecular weight for both trimer and monomer. SPR analyses revealed expected binding with CD4 and multiple broadly neutralizing antibodies. These trimers have markedly different antigenic properties than those of monomeric gp120s derived from the same sequences. They induce potent, cross-clade neutralizing antibody responses with titers substantially higher than those elicited by the corresponding gp120 monomers for a diverse set of both tier 1 and tier 2 viruses. Conclusion We have demonstrated the importance of generating highquality envelope trimers for antigenic and immunogenic studies; furthermore these results highlight the immunologic differences between monomers and high-quality envelope trimers, illustrating important implications for HIV-1 vaccine development and immunogen selection in large clinical trials.

Poly-gamma-D-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea pigs and rabbits (45.8)

Abstract Anthrax is a lethal infectious disease caused by spore-forming Bacillus anthracis. The two major virulence factors of B. anthracis are exotoxin and the poly-gamma-D-glutamic acid (PGA) capsule. The three components of exotoxin, protective antigen (PA), lethal factor and edema factor act in binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of anthrax vaccine. In this study, we report that PA-PGA conjugates induce expression of anti-PA, anti-PGA and toxin neutralizing antibodies in guinea pigs and completely protect guinea pigs against a 50xLD50 challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA 15mer offers passive protection to guinea pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. This work was supported by Korea National Institute of Health Grants (H. H and G. R.).

Antibody response to 146S particle, 12S protein subunit and isolated VP1 polypeptide of foot-and-mouth disease virus type Asia-1

The antibody response to foot-and-mouth disease virus (FMDV) antigens of type Asia-1 in guinea-pigs was studied by micro-serum neutralization test (MSNT) and enzyme-linked immunosorbent assay (ELISA). One inoculation of as little as 1 μg of binary ethyleneimine (BEI)-insctivated 146S virus particles in guinea-pigs elicited enough neutralizing antibodies to protect them against challenge with virulent virus. However, one inoculation of live 146S virus particles elicited higher levels of neutralizing antibodies in guinea-pigs than that of inactivated 146S particles. One inoculation of 12S protein subunits in guinea-pigs elicited only non-neutralizing anti-12S antibodies detected by ELISA. Similarly, non-neutralizing anti-VP1 antibodies were detected by ELISA after one inoculation of VP1. However, multiple inoculations of 12S or VP1 elicited measurable neutralizing antibodies in guinea-pigs that protected them against challenge.

High expression of human cytomegalovirus (HCMV)-gB protein in cells infected with a vaccinia-gB recombinant: the importance of the gB protein in HCMV immunity

The human cytomegalovirus (HCMV), Towne strain, glycoprotein B (gB) gene was cloned into a vaccinia vector (Copenhagen strain) under the control of the H6 early and late vaccinia promoters (Vac-gB recombinant). The gB protein was expressed in a high percentage of the Vac-gB-infected cells throughout the virus replication cycle. Cytosine-arabinoside (ara-C) did not influence the expression of the gB protein early after infection (5 h), but did inhibit it later in viral replication (7–29 h). The Vac-gB recombinant induced HCMV neutralizing antibodies in guinea-pigs. Cells infected with the Vac-gB recombinant absorbed 50–88% of neutralizing activity of human sera obtained from volunteers previously inoculated with the Towne or Toledo strains and from naturally seropositive individuals.

Characterization of two different human cytomegalovirus glycoproteins which are targets for virus neutralizing antibody.

In previous studies we have identified two viral polypeptides detected by murine monoclonal antibodies which neutralize the infectivity of human cytomegalovirus (CMV) AD169. One is an 86,000-Da polypeptide (p86) and the second is a complex of two major coimmunoprecipitating polypeptides of 130,000 and 55,000 Da (p130/55). In this study we have shown that the two viral polypeptides are immunologically unrelated and have distinct peptide cleavage patterns. We have characterized these polypeptides as glycoproteins and studied their biosynthesis in human embryonic lung cells. The oligosaccharides found on both the p86 and the p130/55 were characterized by endoglycosidase digestion as N-linked high-mannose carbohydrates. Inhibitors of glycosylation were used to further characterize the oligosaccharides. Tunicamycin, which inhibits the biosynthesis of N-linked oligosaccharides on the endoplasmic reticulum, inhibited both the infectivity and biosynthesis of the p86 and p130/55. The underglycosylated forms in tunicamycin-treated cultures could be detected only under conditions of pulse-labeling with L-[35S]methionine. Monensin, which inhibits the modification of glycoproteins from simple to complex forms in the Golgi, reduced viral infectivity at concentrations which had no effect on viral protein synthesis, but did not alter the apparent molecular weight of either the p86 or the p130/55. The oligosaccharides were critical for the in vitro immunologic reactivity of the p86 in immunoblots. However, endoglycosidase F-treated p86 was comparable to the native form in inducing virus neutralizing antibody in guinea pigs. Endoglycosidase F-treated p130/55 retained its ability to bind antibody in Western blots.

The structural proteins of the autonomous parvovirus feline panleukopenia virus.

Approximately 80% of the genome of feline panleukopenia virus was cloned into the plasmid pBR322. The entire 3943 nucleotide sequence of the cloned portion of FPV was determined. This DNA includes the gene which codes for the structural proteins of the virus. Portions of this gene were expressed in E. coli as fusion proteins with bacterial proteins. Some of the fusion proteins were capable of raising neutralizing antibodies in guinea pigs. Through the use of deletion mapping, monoclonal antibodies, and synthetic peptides, attempts were made to localize the portion of the protein responsible for raising these antibodies.

Rhinoviruses: Kinetics of ultraviolet inactivation and effects of UV and heat on immunogenicity

The kinetics of ultraviolet inactivation for two human rhinoviruses and poliovirus were compared. No major differences in the rates of ultraviolet inactivation were detectable. All viral preparations inactivated by ultraviolet irradiation induced neutralizing antibody in guinea pigs. In contrast, when guinea pigs were immunized with a heat inactivated rhinovirus preparation, little or no neutralizing antibody was elicited. Immune electron microscopy of the heated rhinovirus preparations revealed the presence of particles resembling empty capsids. These results suggest that rhinoviruses and enteroviruses are affected in a similar manner when subjected to ultraviolet or heat inactivation.

Foot-and-Mouth Disease Virus Immunogenic Capsid Protein VP<sub>T</sub>: N-Terminal Sequences and Immunogenic Peptides Obtained by CNBr and Tryptic Cleavages

The immunogenic capsid protein (VPT), circa 30 kiladaltons (kd), of foot-and-mouth disease virus was examined for (i) its ability to induce neutralizing antibody in guinea pigs after chemical modifications and CNBr or tryptic cleavages and (ii) N-terminal amino sequence homology across three virus types. The immunogenicity of VPT was inactivated by glutaraldehyde treatment, carboxymethylation and maleylation or citraconylation. However, de-citraconylation restored part of the lost activity. Cleavage of type A12 VPT with CNBr produced an immunogenic peptide of circa 13 kd. A slightly larger (ca. 16 kd) immunogenic doublet, VPTab, was obtained by tyrptic cleavage of VPT in the virion. Sequence homologies of circa 85% were found between the first 26 amino acids at the N-terminus of VP chains from virus types A12 strain 119 (A12), C3 Resende (C3R) and O1 Brugge (O1B).

Dissection of vesicular stomatitis virus into the infective ribonucleoprotein and immunizing components.

SUMMARY Treatment of the infective component of vesicular stomatitis virus with Nonidet P40 produces an infective skeleton-like structure which has the shape and approximate size of the intact virus particle. The infectivity of the skeleton is enhanced 100 to 1000 fold by mixing with DEAE-dextran. The skeleton lacks the outer envelope and fringe structure and in consequence does not produce neutralizing antibodies in guinea pigs. The density of the skeleton is 1·22 g./ml. in potassium tartrate gradients compared with 1·14 g./ml. for the virus. Sodium deoxycholate removes protein from the skeleton and releases the filamentous ribonucleoprotein in an infective form. As with the skeleton, the infectivity of the ribonucleoprotein is enhanced by DEAE-dextran. Ribonuclease has no effect on the ribonucleoprotein but trypsin destroys its infectivity. The ribonucleoprotein has a density of 1·22 g./ml. in tartrate gradients, sediments at about 140 s in sucrose gradients and does not produce neutralizing antibodies in guinea-pigs.

Stability of purified foot and mouth disease virus after freeze-drying and storage

Summary Purified type A, strain 119, foot and mouth disease virus was subjected to the stresses of freezing, thawing, drying, and subsequent storage at various temperatures. Degradation of the virus began with the initial freezing and thawing, continued through the drying process, and after 4 weeks of storage had arrived at a point where the remaining virus had little ability to produce plaques in monolayers of cultures of calf kidney cells. No stressed purified virus was capable of inducing detectable viral neutralizing antibodies in guinea pigs because of the degraded or denatured state of the virus. A lack of infectivity of the virus for guinea pigs was probably due to nonadaptation to the species. Freezing, thawing, drying, and storage of the purified virus in the range of −80 to 37°C was not conducive to preserving infectivity of the virus. Such preparations when stored should be preserved frozen at the temperature of liquid nitrogen (−196°).