Production Of Virus-neutralizing Antibodies Research Articles

АНТИГЕННЫЙ И ГЕНЕТИЧЕСКИЙ СОСТАВ РЕСПИРАТОРНО-СИНЦИТИАЛЬНОГО ВИРУСА КРУПНОГО РОГАТОГО СКОТА

The antigenic and genetic composition of bovine respiratory syncytial virus has been studied. It was found that among the structural proteins of RSV in cattle, the most significant in relation to the formation of immunity are proteins F and G, which cause the production of virus-neutralizing antibodies, and the F protein additionally stimulates the formation of CD8 + cytotoxic T cells, which has a reducing impact on the viral replication, and recovery of animals.

Lumpy Skin Disease Virus with Four Knocked Out Genes Was Attenuated In Vivo and Protects Cattle from Infection.

Vaccination with live attenuated vaccines is a key element in the prevention of lumpy skin disease. The mechanism of virus attenuation by long-term passaging in sensitive systems remains unclear. Targeted inactivation of virulence genes is the most promising way to obtain attenuated viruses. Four virulence genes in the genome of the lumpy skin disease virus (LSDV) Dermatitis nodulares/2016/Atyrau/KZ were sequentially knocked out by homologous recombination under conditions of temporary dominant selection. The recombinant LSDV Atyrau-5BJN(IL18) with a knockout of the LSDV005, LSDV008, LSDV066 and LSDV142 genes remained genetically stable for ten passages and efficiently replicated in cells of lamb testicles, saiga kidney and bovine kidney. In vivo experiments with cattle have shown that injection of the LSDV Atyrau-5BJN(IL18) at a high dose does not cause disease in animals or other deviations from the physiological norm. Immunization of cattle with the LSDV Atyrau-5BJN(IL18) induced the production of virus-neutralizing antibodies in titers of 4–5 log2. The challenge did not cause disease in immunized animals. The knockout of four virulence genes resulted in attenuation of the virulent LSDV without loss of immunogenicity. The recombinant LSDV Atyrau-5BJN(IL18) is safe for clinical use, immunogenic and protects animals from infection with the virulent LSDV.

Effectiveness and safety of COVID-19 vaccines in patients with diabetes as a factor for vaccine hesitancy.

Diabetes mellitus is one of the most common comorbid conditions encountered in patients with severe acute respiratory syndrome coronavirus 2 infection accompanied by significantly increased mortality, prolonged hospital stay, and requirement of invasive mechanical ventilation. This review aims to present the effectiveness and safety profile of available coronavirus disease 2019 (COVID-19) vaccines in people with diabetes as a potential cause of hesitancy for vaccination. Data from published research proves a robust immune response following immunization for COVID-19 in diabetic patients with substantial production of virus-neutralizing antibodies; however, the observed immune response was unequivocally weaker than that in individuals without diabetes. This observation was further enhanced by the findings that worse glycemic control was associated with more suppressed antibody production. In contrast, individuals with optimal glycemic control performed similarly to healthy controls. In addition to the need for strict glucose monitoring and adequate diabetes treatment, those findings reinforce the concept of diabetes-induced secondary immune deficiency and necessitate the application of booster doses to diabetic patients with priority. Nevertheless, after vaccination, reported adverse events were not different from those in the general population. No increase in severe adverse events was documented. While single case reports detected transient increases in blood glucose post-vaccination, more extensive trials could not replicate such a relationship.

Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS116-42 antigen.

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116–42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116–42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines.

Immunogenic properties of SARS-CoV-2 inactivated by ultraviolet light.

Vaccination against COVID-19 is the most effective method of controlling the spread of SARS-CoV-2 and reducing mortality from this disease. The development of vaccines with high protective activity against a wide range of SARS-CoV-2 antigenic variants remains relevant. In this regard, evaluation of the effectiveness of physical methods of virus inactivation, such as ultraviolet irradiation (UV) of the virus stock, remains relevant. This study demonstrates that the UV treatment of SARS-CoV-2 completely inactivates its infectivity while preserving its morphology, antigenic properties, and ability to induce the production of virus-neutralizing antibodies in mice through immunization. Thus, the UV inactivation of SARS-CoV-2 makes it possible to obtain viral material similar in its antigenic and immunogenic properties to the native antigen, which can be used both for the development of diagnostic test systems and for the development of an inactivated vaccine against COVID-19.

Hybrid Proteins with Short Conformational Epitopes of the Receptor-Binding Domain of SARS-CoV-2 Spike Protein Promote Production of Virus-Neutralizing Antibodies When Used for Immunization

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein–protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.

A Novel Orf Virus D1701-VrV-Based Dengue Virus (DENV) Vaccine Candidate Expressing HLA-Specific T Cell Epitopes: A Proof-of-Concept Study.

Although dengue virus (DENV) affects almost half of the world’s population there are neither preventive treatments nor any long-lasting and protective vaccines available at this time. The complexity of the protective immune response to DENV is still not fully understood. The most advanced vaccine candidates focus specifically on humoral immune responses and the production of virus-neutralizing antibodies. However, results from several recent studies have revealed the protective role of T cells in the immune response to DENV. Hence, in this study, we generated a novel and potent DENV vaccine candidate based on an Orf virus (ORFV, genus Parapoxvirus) vector platform engineered to encode five highly conserved or cross-reactive DENV human leukocyte antigen (HLA)-A*02- or HLA-B*07-restricted epitopes as minigenes (ORFV-DENV). We showed that ORFV-DENV facilitates the in vitro priming of CD8+ T cells from healthy blood donors based on responses to each of the encoded immunogenic peptides. Moreover, we demonstrated that peripheral blood mononuclear cells isolated from clinically confirmed DENV-positive donors stimulated with ORFV-DENV generate cytotoxic T cell responses to at least three of the expressed DENV peptides. Finally, we showed that ORFV-DENV could activate CD8+ T cells isolated from donors who had recovered from Zika virus (ZIKV) infection. ZIKV belongs to the same virus family (Flaviviridae) and has epitope sequences that are homologous to those of DENV. We found that highly conserved HLA-B*07-restricted ZIKV and DENV epitopes induced functional CD8+ T cell responses in PBMCs isolated from confirmed ZIKV-positive donors. In summary, this proof-of-concept study characterizes a promising new ORFV D1701-VrV-based DENV vaccine candidate that induces broad and functional epitope-specific CD8+ T cell responses.

Inactivated marked vaccine against Aujeszky’s disease

The article presents data on the results of assessing the intensity and duration of the immune response in piglets to the introduction of inactivated marked vaccine against Aujeszky’s disease in comparison with similar indicators for immunization with live labeled and inactivated unlabeled vaccines. Immunization with an inactivated marked vaccine induced the production of virus neutralizing antibodies at a high level.

Porcine circovirus 2 capsid protein produced in N. benthamiana forms virus-like particles that elicit production of virus-neutralizing antibodies in guinea pigs

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

SummaryBackgroundThe spread of SARS-CoV-2 has caused a worldwide pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection and may enable the relaxation of social-distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection; therefore, it is desirable that any new vaccine candidates elicit a robust immune response in older adults.MethodsHere, we use in-depth immunophenotyping to characterize the innate and adaptive immune response induced upon intramuscular administration of the adenoviral vectored ChAdOx1 nCoV-19 (AZD-1222) COVID-19 vaccine candidate in mice.FindingsA single vaccination generates spike-specific Th1 cells, Th1-like Foxp3+ regulatory T cells, polyfunctional spike-specific CD8+ T cells. and granzyme-B-producing CD8 effectors. Spike-specific IgG and IgM are generated from both the early extrafollicular antibody response and the T follicular helper cell-supported germinal center reaction, which is associated with the production of virus-neutralizing antibodies. A single dose of this vaccine generated a similar type of immune response in aged mice but of a reduced magnitude than in younger mice. We report that a second dose enhances the immune response to this vaccine in aged mice.ConclusionsThis study shows that ChAdOx1 nCoV-19 induces both cellular and humoral immunity in adult and aged mice and suggests a prime-boost strategy is a rational approach to enhance immunogenicity in older persons.FundingThis study was supported by BBSRC, Lister institute of Preventative Medicine, EPSRC VaxHub, and Innovate UK.

Increased antiviral response in circulating lymphocytes from hypogammaglobulinemia patients.

B cells play a crucial role during rhinovirus (RV) infections by production of virus-neutralizing antibodies. A main feature of common variable immunodeficiency (CVID) is hypogammaglobulinemia (HG). HG patients have severely reduced levels of antibody-producing B cells and suffer from prolonged virus infections. Here, we addressed whether antiviral response of peripheral blood lymphocytes differs between HG patients and healthy individuals during natural RV infection. Using fluorescence-activated cell sorting, B-cell subsets were analyzed. Simultaneously, CD19+B cells, CD14+monocytes, and CD3+T cells were sorted from frozen peripheral blood mononuclear cells from 11 RV-infected hypogammaglobulinemia patients, 7 RV-infected control subjects, and 14 noninfected control subjects. Real-time PCR was used to study expression of antiviral genes. A pan-RV PCR was used to detect RV genome in all samples. In HG patients, total B-cell numbers, as well as IgA+and IgG+switched memory B cells, were reduced while naïve B cells and T cells were increased. STAT1 expression was increased in HG patients compared to controls in all lymphocyte subsets analyzed. The expression of antiviral genes IFITM1 and MX1 correlated with STAT1 expression in B cells and monocytes. RV RNA was found in 88.9% of monocytes from infected HG patients, 85.7% of monocytes from infected controls, and 7.1% of monocytes from uninfected controls. We demonstrate an increased antiviral response in B cells and monocytes in HG patients and their correlation with STAT1 expression. Monocytes of infected HG patients and infected non-HG controls carry RV RNA.

Влияние адъювантов различных групп на иммуногенные свойства кандидатных вакцин против геморрагической лихорадки с почечным синдромом

Effects of different classes of adjuvants (aluminum hydroxide, spherical protein particles, thermolabile В protein, low-endotoxic pyrogen-free lipopolysaccharide) have been studied in order to enhance the immunogenicity of inactivated vaccines against hemorrhagic fever with renal syndrome. The Puumala virus-based monovaccine and multivalent vaccine based on Puumala, Hantaan and Sochi viruses were analyzed, and BALB/c mice were used as an animal model. It was shown that low-endotoxic pyrogen-free lipopolysaccharide stimulated the production of virus-neutralizing antibodies and increased the vaccine stability during storage, which allows to reduce the antigenic load of the vaccine. Aluminum hydroxide slightly increased the production of T-cells immune response mediators and did not affect the neutralizing antibodies induction and vaccine stability. Despite the adjuvant effect, it was shown that spherical protein particles and thermolabile В protein were unacceptable for vaccines administered to humans due to the high protein load and toxic effects, respectively. hantavirases, hemorrhagic fever with renal syndrome, inactivated vaccines, adjuvants, immune response The authors are grateful to Dr. O.V. Karpova (Department of Virology, Moscow State University) for providing the preparation of spherical particles, and to Dr. A.N. Noskov (Gamaleya Research Institute of Epidemiology and Microbiology, Moscow) for providing the preparation of the thermolabile enterotoxin B-subunit.

Peste des Petits Ruminants Virus-Like Particles Induce a Potent Humoral and Cellular Immune Response in Goats.

Peste des petits ruminants is a highly contagious acute or subacute disease of small ruminants caused by the peste des petits ruminants virus (PPRV), and it is responsible for significant economic losses in animal husbandry. Vaccination represents the most effective means of controlling this disease, with virus-like particle (VLP) vaccines offering promising vaccine candidates. In this study, a PPRV VLP-based vaccine was developed using a baculovirus expression system, allowing for the simultaneous expression of the PPRV matrix (M), hemagglutinin (H), fusion (F) and nucleocapsid (N) proteins in insect cells. Immunization of mice and goats with PPRV VLPs elicited a robust neutralization response and a potent cellular immune response. Mouse studies demonstrated that VLPs induced a more robust IFN-γ response in CD4+ and CD8+ T cells than PPRV Nigeria 75/1 and recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. In addition, PPRV VLPs induced a strong Th1 class response in mice, as indicated by a high IgG2a to IgG1 ratio. Goat studies demonstrated that PPRV VLPs can induce the production of antibodies specific for F and H proteins and can also stimulate the production of virus neutralizing antibodies to the same magnitude as the PPRV Nigeria 75/1 vaccine. Higher amounts of IFN-γ in VLP-immunized animal serum suggested that VLPs also elicited a cellular immune response in goats. These results demonstrated that VLPs elicit a potent immune response against PPRV infection in small ruminants, making PPRV VLPs a potential candidate for PPRV vaccine development.

Deficient Incorporation of Rabies Virus Glycoprotein into Virions Enhances Virus-Induced Immune Evasion and Viral Pathogenicity.

Previous studies have shown that wild-type (wt) rabies virus (RABV) evades the host immune response by restricting expression of glycoprotein (G), which blocks activation of dendritic cells (DCs) and induces production of virus-neutralizing antibodies (VNAs). In the present study, wt RABVs not only restricted G expression but also reduced incorporation of G into mature virions compared with laboratory-adapted viruses. A recombinant RABV expressing triple G was used to further determine whether G expression relates to incorporation. The recombinant virus showed higher expression and incorporation of G and activated more DCs than the virus that expressed a single copy of G. Removal of G from viruses using subtilisin or Dithiothreitol (DTT)/ Nonidet P-40 (NP40) almost completely abolishes DC activation and VNA production. Consequently, these G-depleted viruses cause lethal infection in mice. Thus, wt RABVs can subvert DC-induced antiviral immune response and maintain pathogenicity by decreasing G expression in infected cells and G incorporation into virions.

Different mechanisms of the protection against influenza A infection mediated by broadly reactive HA2-specific antibodies.

Influenza Aviruses (IAVs) cause yearly repeating infections in humans. The current vaccination approach is based on the production of virus-neutralizing antibodies. Virus-neutralizing antibodies, however, are closely strain-specific due to the IAV variability. Therefore, antibodies produced during the previous influenza season do not provide sufficient protection against new infection, and, hence, annual revaccination is needed. The utilization of the influenza conserved stem domain of hemagglutinin (HA), the HA2 gp, led to a new vaccine design based on cross-reactive cellular and especially humoral immune responses represented by HA2-specific antibodies. The HA2-specific antibodies exhibit cross-reactivity with HA2 gp within one subtype or even among subtypes and play a role in protective immunity against influenza infection. There are several elimination mechanisms of viral replication mediated by HA2-specific antibodies. After recognition of the epitope, they prevent the conformational rearrangement of HA or the insertion of the fusion protein into the endosomal membrane and, consequently, the fusion pore formation. In this case, no release of viral genetic information into the target cell is enabled and virus cannot replicate. The HA2-specific antibodies are involved in the elimination of pathogen via the Fc fragment by activation of the cytotoxic mechanisms of innate immunity as are the antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), or complement-dependent cytotoxicity (CDC), resulting in virus elimination and earlier recovery of the host from the infection. Though the protective effect of HA2-specific antibodies on the course of IAV infection was shown, few cases of worsening of IAV infection mediated by HA2-specific antibodies have been described. The identification of antigenic epitopes on HA2 gp that induce antibodies with such deteriorating effect on influenza infection can help to eliminate the unsuitable epitopes of HA2 gp as immunogens during the design of heteroprotective vaccine against influenza and can remove the side effects linked with the observations mentioned above. Keywords: influenza A virus; HA2 stem domain of hemagglutinin; immunization strategies; HA2-specific antibodies.

A Link Between a Common Mutation in CFTR and Impaired Innate and Adaptive Viral Defense.

Acute respiratory virus infections predispose the cystic fibrosis (CF) lung to chronic bacterial colonization, which contributes to high mortality. For reasons unknown, respiratory virus infections have a prolonged duration in CF. Here, we demonstrate that mice carrying the most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutation in humans, ΔF508, show increased morbidity and mortality following infection with a common human enterovirus. ΔF508 mice demonstrated impaired viral clearance, a slower type I interferon response and delayed production of virus-neutralizing antibodies. While the ΔF508 mice had a normal immune cell repertoire, unchanged serum immunoglobulin concentrations and an intact immune response to a T-cell-independent antigen, their response to a T-cell-dependent antigen was significantly delayed. Our studies reveal a novel function for CFTR in antiviral immunity and demonstrate that the ΔF508 mutation in cftr is coupled to an impaired adaptive immune response. This important insight could open up new approaches for patient care and treatment.

Neutralizing Antibody Responses to Viral Infections Are Linked to the Non-classical MHC Class II Gene H2-Ob

Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Protective effect and antibody response of DNA vaccine against salmonid alphavirus 3 (SAV3) in Atlantic salmon.

This work reports the effect of two DNA vaccines against salmonid alphavirus 3 (SAV3) in Atlantic salmon. Presmolts were vaccinated by intramuscular injection of plasmids encoding the SAV3 structural polyprotein C-E3-E2-6K-E2 (pCSP), E2 only (pE2), or plasmid without insert (pcDNA3.3). E2 is expressed at the surface of cells transfected with pCSP and internally in cells transfected with pE2. A commercial vaccine based on inactivated SAV (NCPD) was used for comparison. At 10weeks post-vaccination, only fish vaccinated with pCSP showed antibody against E2 and virus-neutralizing activity. Vaccinated fish were infected with SAV3 to determine protection by virus quantitation in serum after 7days and scoring of pathological changes after 21days. Fish vaccinated with both pCSP and NCPD vaccines showed significant virus reduction in serum, while fish vaccinated with pE2 did not. All fish vaccinated with pcDNA3.3 and pE2 showed pathological changes in organs typical of PD, 60% of fish vaccinated with NCPD showed PD pathology, while fish vaccinated with pCSP did not show PD pathology. Taken together, DNA vaccination with pCSP provided strong protection for salmon against SAV3 infection, which in part may be due to production of virus-neutralizing antibodies.

Encapsulation of Adenovirus BMP2-Transduced Cells with PEGDA Hydrogels Allows Bone Formation in the Presence of Immune Response

Gene therapy approaches have been difficult to implement due to pre-existing immunity against the virus used for delivery. To circumvent this problem, a cell-based approach was developed that avoided the use of free virus within the animal. However, even cells transduced in vitro with E1- to E3-deleted adenovirus encoding bone morphogenetic protein 2 (AdBMP2) resulted in the production of virus-neutralizing antibodies in mice. Furthermore, when mice received an intramuscular injection of nonencoding adenovirus (AdEmpty)-transduced cells, AdBMP2-transduced cells were unable to launch bone formation when an intramuscular injection of these BMP2-producing cells was delivered 1 week later. This phenomenon was not observed in NOD/SCID mice, and could be overcome in C57BL/6 mice by encapsulating the adenovirus-transduced cells in a nondegradable hydrogel poly(ethylene glycol) diacrylate (PEGDA). Data collectively suggest that PEGDA hydrogel encapsulation of AdBMP2-transduced cells prevents pre-existing immunity from suppressing BMP2-induced bone formation.

Clinical and immunological assessment of therapeutic immunization with a subunit vaccine for recurrent ocular canine herpesvirus-1 infection in dogs

Latent canine herpesvirus-1 (CHV-1) infections are common in domestic dogs and reactivation of latent virus may be associated with recurrent ocular disease. The objectives of the present study were to evaluate the ability of a subunit CHV-1 vaccine to stimulate peripheral CHV-1 specific immunity and prevent recurrent CHV-1 ocular disease and viral shedding. Mature dogs with experimentally-induced latent CHV-1 infection received a 2-dose CHV-1 vaccine series. Recurrent ocular CHV-1 infection was induced by corticosteroid administration in the prevaccinal, short-term postvaccinal (2 weeks post-vaccination), and long-term postvacccinal (34 weeks post-vaccination) periods. Immunological, virological, and clinical parameters were evaluated during each study period. Quantitative assessment of peripheral immunity included lymphocyte immunophenotyping, proliferation response, and interferon-γ production; and CHV-1 virus neutralizing antibody production. In the present study, vaccination did not prevent development of ocular disease and viral shedding; however, there was a significant decrease in clinical ocular disease scores in the short-term postvaccinal period. Significant alterations in peripheral immunity detected in the dogs during the short-term and long-term postvaccinal periods included increased T and B lymphocyte subpopulation percentage distributions, increased lymphocyte expression of major histocompatibility complex class I and II, increased CHV-1 virus neutralizing antibody titers, decreased lymphocyte proliferation, and decreased interferon-γ production. Vaccination of latently infected mature dogs with the selected subunit CHV-1 vaccine was not effective in preventing recurrent ocular CHV-1 infection and viral shedding induced by corticosteroid administration. The vaccine did induce long-term CHV-1 specific immunity and may decrease the severity of clinical ocular disease in the immediate postvaccinal period.