High recallability of memory B cells requires ZFP318-dependent transcriptional regulation of mitochondrial function.

Expression of the transcriptional regulator ZFP318 is induced in germinal center (GC)-exiting memory B cell precursors and memory B cells (MBCs). Using a conditional ZFP318 fluorescence reporter that also enables ablation of ZFP318-expressing cells, we found that ZFP318-expressing MBCs were highly enriched with GC-derived cells. Although ZFP318-expressing MBCs constituted only a minority of the antigen-specific MBC compartment, their ablation severely impaired recall responses. Deletion of Zfp318 did not alter the magnitude of primary responses but markedly reduced MBC participation in recall. CD40 ligation promoted Zfp318 expression, whereas B cell receptor (BCR) signaling was inhibitory. Enforced ZFP318 expression enhanced recall performance of MBCs that otherwise responded poorly. ZFP318-deficient MBCs expressed less mitochondrial genes, had structurally compromised mitochondria, and were susceptible to reactivation-induced cell death. The abundance of ZFP318-expressing MBCs, instead of the number of antigen-specific MBCs, correlated with the potency of prime-boost vaccination. Therefore, ZFP318 controls the MBC recallability and represents a quality checkpoint of humoral immune memory.

Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Pre-existing humoral immunity and CD4+ T cell response correlate with cross-reactivity against SARS-CoV-2 Omicron subvariants after heterologous prime-boost vaccination.

BACKGROUND: Information regarding the heterologous prime-boost COVID vaccination has been fully elucidated. The study aimed to evaluate both humoral, cellular immunity and cross-reactivity against variants after heterologous vaccination. METHODS: We recruited healthcare workers previously primed with Oxford/AstraZeneca ChAdOx1-S vaccines and boosted with Moderna mRNA-1273 vaccine boost to evaluate the immunological response. Assay used: anti-spike RBD antibody, surrogate virus neutralizing antibody and interferon-γ release assay. RESULTS: All participants exhibited higher humoral and cellular immune response after the booster regardless of prior antibody level, but those with higher antibody level demonstrated stronger booster response, especially against omicron BA.1 and BA.2 variants. The pre-booster IFN-γ release by CD4+ T cells correlates with post-booster neutralizing antibody against BA.1 and BA.2 variant after adjustment with age and gender. CONCLUSIONS: A heterologous mRNA boost is highly immunogenic. The pre-existing neutralizing antibody level and CD4+ T cells response correlates with post-booster neutralization reactivity against the Omicron variant.

Transient T Cell Expansion, Activation, and Proliferation in Therapeutically Vaccinated Simian Immunodeficiency Virus-Positive Macaques Treated with N-803.

Vaccine strategies aimed at eliciting human immunodeficiency virus (HIV)-specific CD8+ T cells are one major target of interest in HIV functional cure strategies. We hypothesized that CD8+ T cells elicited by therapeutic vaccination during antiretroviral therapy (ART) would be recalled and boosted by treatment with the interleukin 15 (IL-15) superagonist N-803 after ART discontinuation. We intravenously immunized four simian immunodeficiency virus-positive (SIV+) Mauritian cynomolgus macaques receiving ART with vesicular stomatitis virus (VSV), modified vaccinia virus Ankara strain (MVA), and recombinant adenovirus serotype 5 (rAd-5) vectors all expressing SIVmac239 Gag. Immediately after ART cessation, these animals received three doses of N-803. Four control animals received no vaccines or N-803. The vaccine regimen generated a high-magnitude response involving Gag-specific CD8+ T cells that were proliferative and biased toward an effector memory phenotype. We then compared cells elicited by vaccination (Gag specific) to cells elicited by SIV infection and unaffected by vaccination (Nef specific). We found that N-803 treatment enhanced the frequencies of both bulk and proliferating antigen-specific CD8+ T cells elicited by vaccination and the antigen-specific CD8+ T cells elicited by SIV infection. In sum, we demonstrate that a therapeutic heterologous prime-boost-boost (HPBB) vaccine can elicit antigen-specific effector memory CD8+ T cells that are boosted by N-803. IMPORTANCE While antiretroviral therapy (ART) can suppress HIV replication, it is not a cure. It is therefore essential to develop therapeutic strategies to enhance the immune system to better become activated and recognize virus-infected cells. Here, we evaluated a novel therapeutic vaccination strategy delivered to SIV+ Mauritian cynomolgus macaques receiving ART. ART was then discontinued and we delivered an immunotherapeutic agent (N-803) after ART withdrawal with the goal of eliciting and boosting anti-SIV cellular immunity. Immunologic and virologic analysis of peripheral blood and lymph nodes collected from these animals revealed transient boosts in the frequency, activation, proliferation, and memory phenotype of CD4+ and CD8+ T cells following each intervention. Overall, these results are important in educating the field of the transient nature of the immunological responses to this particular therapeutic regimen and the similar effects of N-803 on boosting T cells elicited by vaccination or elicited naturally by infection.

Robust and protective immune responses induced by heterologous prime-boost vaccination with DNA-protein dimeric RBD vaccines for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic posed great impacts on public health. To fight against the pandemic, robust immune responses induced by vaccination are indispensable. Previously, we developed a subunit vaccine adjuvanted by aluminum hydroxide, ZF2001, based on the dimeric tandem-repeat RBD immunogen, which has been approved for clinical use. This dimeric RBD design was also explored as an mRNA vaccine. Both showed potent immunogenicity. In this study, a DNA vaccine candidate encoding RBD-dimer was designed. The humoral and cellular immune responses induced by homologous and heterologous prime-boost approaches with DNA-RBD-dimer and ZF2001 were assessed in mice. Protection efficacy was studied by the SARS-CoV-2 challenge. We found that the DNA-RBD-dimer vaccine was robustly immunogenic. Priming with DNA-RBD-dimer followed by ZF2001 boosting induced higher levels of neutralizing antibodies than homologous vaccination with either DNA-RBD-dimer or ZF2001, elicited polyfunctional cellular immunity with a TH 1-biased polarization, and efficiently protected mice against SARS-CoV-2 infection in the lung. This study demonstrated the robust and protective immune responses induced by the DNA-RBD-dimer candidate and provided a heterologous prime-boost approach with DNA-RBD-dimer and ZF2001.

Comparison of heterologous prime-boost immunization strategies with DNA and recombinant vaccinia virus co-expressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus in pigs.

Vaccination is principally used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. This study investigated immunogenicity and protective efficacy of heterologous prime-boost regimens in pigs, including recombinant DNA and vaccinia virus vectors coexpressing PRRSV European genotype (EU) isolate GP3 and GP5: group A, pVAX1-EU-GP3-GP5 prime and rddVTT-EU-GP3-GP5 boost; group B, rddVTT-EU-GP3-GP5 prime and pVAX1-EU-GP3-GP5 boost; group C, empty vector pVAX1; group D, E3L gene-deleted vaccinia virus E3L- VTT. Vaccine efficacy was tested in an EU-type PRRSV (Lelystad virus strain) challenge pig model based on evaluating PRRSV-specific antibody responses, neutralizing antibodies, cytokines, T lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, clinical symptoms, viremia and tissue virus loads. Plasmid DNA was delivered as chitosan-DNA nanoparticles, and Quil A (Quillaja) was used to increase vaccine efficiency. All piglets were boosted 21 days post the initial inoculation (dpi) and then challenged 14 days later. At 14, 21, 28 and 35 dpi, groups A and B developed significantly higher PRRSV-specific antibody responses compared with control groups C and D. Two weeks after the boost, significant differences in neutralizing antibody and IFN-γ levels were observed between groups A, C, D and B. At 49 dpi, groups A and B had markedly increased peripheral blood CD3+CD4+ T cell levels. Following virus challenge, group A showed viremia, but organ virus loads were lower than those in other groups. Thus, a heterologous prime-boost vaccine regimen (rddVTT-EU-GP3-GP5 prime, pVAX1-EU-GP3-GP5 boost) can improve humoral- and cell-mediated immune responses to provide resistance to EU-type PRRSV infection in vivo.

LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

OBJECTIVES: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. METHODS: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. RESULTS: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. CONCLUSION: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Immunogenicity and safety of heterologous mRNA-1273/MVC-COV1901 vaccination versus homologous mRNA1273 vaccination: A randomized, double-blind controlled study.

BACKGROUND/PURPOSE: MVC-COV1901 is a protein vaccine based on the same SARS-CoV-2 strain used in mRNA vaccine mRNA-1273. Data are lacking on immunogenicity and safety of MVC-COV1901 as heterologous boost for people already received one dose of mRNA-1273. METHODS: This is a randomized, double-blind trial that recruited adults aged 20-70 years who previously received a single dose of mRNA-1273 vaccine and were randomly assigned in a 1:1 ratio to receive a second dose with the homologous vaccine or protein-based MVC-COV1901 8-12 weeks after the first dose. The primary outcome was neutralizing antibody titers in terms of the geometric mean titer (GMT) 14 days after the second dose. Safety was assessed in all participants who received a dose of the study vaccine. The study is registered with ClinicalTrials.gov (NCT05079633). RESULTS: From September 30 to November 5, 2021, 144 participants were enrolled and randomly assigned to the MVC-COV1901 boost group (n = 72) or the mRNA-1273 boost group (n = 72). The neutralizing antibodies on Day 15 and the anti-SARS-CoV-2 IgG titers on Day 15 and 29 of homologous mRNA-1273 were significantly higher than those of heterologous mRNA-1273/MVC-COV1901. Cellular immune responses were comparable in both groups. However, adverse events were much more frequent after the mRNA-1273 boost than after the MVC-COV1901 boost. CONCLUSION: Our results show that heterologous boost with MVC-COV1901 yielded an inferior immunogenicity but significantly fewer adverse events, compared with homologous boost with mRNA-1273. In people experienced severe adverse events after prime dose of mRNA-1273, as well as in periods when the supply of mRNA-1273 is limited, MVC-COV1901 could serve as an acceptable alternative heterologous boost.

The Induction of Antigen 85B-Specific CD8+ T Cells by Recombinant BCG Protects against Mycobacterial Infection in Mice.

Mycobacterium tuberculosis (Mtb) infection remains a major health problem worldwide. Although the Bacillus Calmette-Guérin (BCG) vaccine is the most widely used vaccination for preventing tuberculosis (TB), its efficacy is limited. We previously developed a new recombinant BCG (rBCG)-based vaccine encoding the Ag85B protein of M. kansasii (Mkan85B), termed rBCG-Mkan85B, and its administration is followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). Previously, we identified MHC-I (H2-Kd)-restricted epitopes that highly cross-react with those of Mtb in BALB/c (H2d) and CB6F1 (H2b/d) mice. We also reported that the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol protected CB6F1 mice against M. kansasii infection. In this study, to investigate the protective effect of our novel rBCG against Mtb infection, CB6F1 mice were either left unimmunized or immunized with the BCG, rBCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B vaccine for 10 weeks prior to inhalation exposure to the virulent Mtb Erdman strain for another 6 weeks. Compared with the BCG and rBCG-Mkan85B vaccinations, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination protocol significantly reduced the numbers of pulmonary colony-forming units (CFUs). Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime-boost vaccination induced antigen-specific polyfunctional CD4+ and CD8+ T cells. These results suggest that CD8+ T-cell immunity to immunodominant epitopes of Mtb is enhanced by rBCG vector-based immunization. Thus, rBCG vector-based vaccinations may overcome the limited ability of the current BCG vaccine to elicit TB immunity.

Heterologous DNA Prime- Subunit Protein Boost with Chikungunya Virus E2 Induces Neutralizing Antibodies and Cellular-Mediated Immunity.

Chikungunya virus (CHIKV) has become a significant public health concern due to the increasing number of outbreaks worldwide and the associated comorbidities. Despite substantial efforts, there is no specific treatment or licensed vaccine against CHIKV to date. The E2 glycoprotein of CHIKV is a promising vaccine candidate as it is a major target of neutralizing antibodies during infection. In this study, we evaluated the immunogenicity of two DNA vaccines (a non-targeted and a dendritic cell-targeted vaccine) encoding a consensus sequence of E2CHIKV and a recombinant protein (E2*CHIKV). Mice were immunized with different homologous and heterologous DNAprime-E2* protein boost strategies, and the specific humoral and cellular immune responses were accessed. We found that mice immunized with heterologous non-targeted DNA prime- E2*CHIKV protein boost developed high levels of neutralizing antibodies, as well as specific IFN-γ producing cells and polyfunctional CD4+ and CD8+ T cells. We also identified 14 potential epitopes along the E2CHIKV protein. Furthermore, immunization with recombinant E2*CHIKV combined with the adjuvant AS03 presented the highest humoral response with neutralizing capacity. Finally, we show that the heterologous prime-boost strategy with the non-targeted pVAX-E2 DNA vaccine as the prime followed by E2* protein + AS03 boost is a promising combination to elicit a broad humoral and cellular immune response. Together, our data highlights the importance of E2CHIKV for the development of a CHIKV vaccine.

Evaluation of Immune Response to Mucosal Immunization with an Oral Probiotic-Based Vaccine in Mice: Potential for Prime-Boost Immunization against SARS-CoV-2.

Following the conclusion of the COVID-19 pandemic, the persistent genetic variability in the virus and its ongoing circulation within the global population necessitate the enhancement of existing preventive vaccines and the development of novel ones. A while back, we engineered an orally administered probiotic-based vaccine, L3-SARS, by integrating a gene fragment that encodes the spike protein S of the SARS-CoV-2 virus into the genome of the probiotic strain E. faecium L3, inducing the expression of viral antigen on the surface of bacteria. Previous studies demonstrated the efficacy of this vaccine candidate in providing protection against the virus in Syrian hamsters. In this present study, utilizing laboratory mice, we assess the immune response subsequent to immunization via the gastrointestinal mucosa and discuss its potential as an initial phase in a two-stage vaccination strategy. Our findings indicate that the oral administration of L3-SARS elicits an adaptive immune response in mice. Pre-immunization with L3-SARS enhances and prolongs the humoral immune response following a single subcutaneous immunization with a recombinant S-protein analogous to the S-insert of the coronavirus in Enterococcus faecium L3.

Protection Induced by Oral Vaccination with a Recombinant Yersinia pseudotuberculosis Delivering Yersinia pestis LcrV and F1 Antigens in Mice and Rats against Pneumonic Plague.

A newly attenuated Yersinia pseudotuberculosis strain (designated Yptb1) with triple mutation Δasd ΔyopK ΔyopJ and chromosomal insertion of the Y. pestis caf1R-caf1M-caf1A-caf1 operon was constructed as a live vaccine platform. Yptb1 tailored with an Asd+ plasmid (pYA5199) (designated Yptb1[pYA5199]) simultaneously delivers Y. pestis LcrV and F1. The attenuated Yptb1(pYA5199) localized in the Peyer's patches, lung, spleen, and liver for a few weeks after oral immunization without causing any disease symptoms in immunized rodents. An oral prime-boost Yptb1(pYA5199) immunization stimulated potent antibody responses to LcrV, F1, and Y. pestis whole-cell lysate (YPL) in Swiss Webster mice and Brown Norway rats. The prime-boost Yptb1(pYA5199) immunization induced higher antigen-specific humoral and cellular immune responses in mice than a single immunization did, and it provided complete short-term and long-term protection against a high dose of intranasal Y. pestis challenge in mice. Moreover, the prime-boost immunization afforded substantial protection for Brown Norway rats against an aerosolized Y. pestis challenge. Our study highlights that Yptb1(pYA5199) has high potential as an oral vaccine candidate against pneumonic plague.

A Live-Attenuated Zika Virus Vaccine with High Production Capacity Confers Effective Protection in Neonatal Mice.

Zika virus (ZIKV) infection during pregnancy has been linked to congenital abnormalities, such as microcephaly in infants. An efficacious vaccine is desirable for preventing the potential recurrence of ZIKV epidemic. Here, we report the generation of an attenuated ZIKV (rGZ02a) that has sharply decreased virulence in mice but grows to high titers in Vero cells, a widely approved cell line for manufacturing human vaccines. Compared to the wild-type ZIKV (GZ02) and a plasmid-launched rGZ02p, rGZ02a has 3 unique amino acid alterations in the envelope (E, S304F), nonstructural protein 1 (NS1, R103K), and NS5 (W637R). rGZ02a is more sensitive to type I interferon than GZ02 and rGZ02p, and causes no severe neurological disorders in either wild-type neonatal C57BL/6 mice or type I interferon receptor knockout (Ifnar1-/-) C57BL/6 mice. Immunization with rGZ02a elicits robust inhibitory antibody responses with a certain long-term durability. Neonates born to the immunized dams are effectively protected against ZIKV-caused neurological disorders and brain damage. rGZ02a as a booster vaccine greatly improves the protective immunity primed by Ad2-prME, an adenovirus-vectored vaccine expressing ZIKV prM and E proteins. Our results illustrate that rGZ02a-induced maternal immunity can be transferred to the neonates and confer effective protection. Hence, rGZ02a may be developed as an alternative live-attenuated vaccine and warrants further evaluation. IMPORTANCE Zika virus (ZIKV), a mosquito-borne flavivirus that has caused global outbreaks since 2013, is associated with severe neurological disorders, such as Guillian-Barré syndrome in adults and microcephaly in infants. The ZIKV epidemic has gradually subsided, but a safe and effective vaccine is still desirable to prevent its potential recurrence, especially in countries of endemicity with competent mosquito vectors. Here, we describe a novel live-attenuated ZIKV, rGZ02a, that carries 3 unique amino acid alterations compared to the wild-type GZ02 and a plasmid-launched rGZ02p. The growth capacity of rGZ02a is comparable to GZ02 in Vero cells, but the pathogenicity is significantly attenuated in two mice models. Immunization with rGZ02a elicits robust inhibitory antibody responses in the dams and effectively protects their offspring against ZIKV disease. Importantly, in a heterologous prime-boost regimen, rGZ02a effectively boosts the protective immunity primed by an adenovirus-vectored vaccine. Thus, rGZ02a is a promising candidate for a live-attenuated ZIKV vaccine.

Alternating 3 different influenza vaccines for swine in Europe for a broader antibody response and protection.

Heterologous prime-boost vaccination with experimental or commercial influenza vaccines has been successful in various animal species. In this study, we have examined the efficacy of alternating 3 different European commercial swine influenza A virus (swIAV) vaccines: the trivalent Respiporc® FLU3 (TIV), the bivalent GRIPORK® (BIV) and the monovalent Respiporc® FLUpan H1N1 (MOV). Five groups of 6 pigs each received 3 vaccinations at 4-6 week intervals in a homologous or heterologous prime-boost regimen. A sixth group served as a mock-vaccinated challenge control. Four weeks after the last vaccination, pigs were challenged intranasally with a European avian-like H1N1 (1C.2.1) swIAV, which was antigenically distinct from the vaccine strains. One heterologous prime-boost group (TIV-BIV-MOV) had higher hemagglutination inhibition (HI) and neuraminidase inhibition antibody responses against a panel of antigenically distinct H1N1, H1N2 and H3N2 IAVs than the other heterologous prime-boost group (BIV-TIV-MOV) and the homologous prime-boost groups (3xTIV; 3xBIV; 3xMOV). Group TIV-BIV-MOV had seroprotective HI titers (≥ 40) against 56% of the tested viruses compared to 33% in group BIV-TIV-MOV and 22-39% in the homologous prime-boost groups. Post-challenge, group TIV-BIV-MOV was the single group with significantly reduced virus titers in all respiratory samples compared to the challenge control group. Our results suggest that the use of different commercial swIAV vaccines for successive vaccinations may result in broader antibody responses and protection than the traditional, homologous prime-boost vaccination regimens. In addition, the order in which the different vaccines are administered seems to affect the breadth of the antibody response and protection.

Replication-Deficient Lymphocytic Choriomeningitis Virus-Vectored Vaccine Candidate for the Induction of T Cell Immunity against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) represents a major burden to global health, and refined vaccines are needed. Replication-deficient lymphocytic choriomeningitis virus (rLCMV)-based vaccine vectors against cytomegalovirus have proven safe for human use and elicited robust T cell responses in a large proportion of vaccine recipients. Here, we developed an rLCMV vaccine expressing the Mtb antigens TB10.4 and Ag85B. In mice, rLCMV elicited high frequencies of polyfunctional Mtb-specific CD8 and CD4 T cell responses. CD8 but not CD4 T cells were efficiently boosted upon vector re-vaccination. High-frequency responses were also observed in neonatally vaccinated mice, and co-administration of rLCMV with Expanded Program of Immunization (EPI) vaccines did not result in substantial reciprocal interference. Importantly, rLCMV immunization significantly reduced the lung Mtb burden upon aerosol challenge, resulting in improved lung ventilation. Protection was associated with increased CD8 T cell recruitment but reduced CD4 T cell infiltration upon Mtb challenge. When combining rLCMV with BCG vaccination in a heterologous prime-boost regimen, responses to the rLCMV-encoded Mtb antigens were further augmented, but protection was not significantly different from rLCMV or BCG vaccination alone. This work suggests that rLCMV may show utility for neonatal and/or adult vaccination efforts against pulmonary tuberculosis.

Murine CD8 T-cell functional avidity is stable in vivo but not in vitro: Independence from homologous prime/boost time interval and antigen density.

It is known that for achieving high affinity antibody responses, vaccines must be optimized for antigen dose/density, and the prime/boost interval should be at least 4 weeks. Similar knowledge is lacking for generating high avidity T-cell responses. The functional avidity (FA) of T cells, describing responsiveness to peptide, is associated with the quality of effector function and the protective capacity in vivo. Despite its importance, the FA is rarely determined in T-cell vaccination studies. We addressed the question whether different time intervals for short-term homologous vaccinations impact the FA of CD8 T-cell responses. Four-week instead of 2-week intervals between priming and boosting with potent subunit vaccines in C57BL/6 mice did not improve FA. Equally, similar FA was observed after vaccination with virus-like particles displaying low versus high antigen densities. Interestingly, FA was stable in vivo but not in vitro, depending on the antigen dose and the time interval since T-cell activation, as observed in murine monoclonal T cells. Our findings suggest dynamic in vivo modulation for equal FA. We conclude that low antigen density vaccines or a minimal 4-week prime/boost interval are not crucial for the T-cell's FA, in contrast to antibody responses.

Priming with DNA Expressing Trimeric HIV V1V2 Alters the Immune Hierarchy Favoring the Development of V2-Specific Antibodies in Rhesus Macaques.

The RV144 vaccine trial revealed a correlation between reduced risk of HIV infection and the level of nonneutralizing-antibody (Ab) responses targeting specific epitopes in the second variable domain (V2) of the HIV gp120 envelope (Env) protein, suggesting this region as a target for vaccine development. To favor induction of V2-specific Abs, we developed a vaccine regimen that included priming with DNA expressing an HIV V1V2 trimeric scaffold immunogen followed by booster immunizations with a combination of DNA and protein in rhesus macaques. Priming vaccination with DNA expressing the HIV recombinant subtype CRF01_AE V1V2 scaffold induced higher and broader V2-specific Ab responses than vaccination with DNA expressing CRF01_AE gp145 Env. Abs recognizing the V2 peptide that was reported as a critical target in RV144 developed only after the priming immunization with V1V2 DNA. The V2-specific Abs showed several nonneutralizing Fc-mediated functions, including ADCP and C1q binding. Importantly, robust V2-specific Abs were maintained upon boosting with gp145 DNA and gp120 protein coimmunization. In conclusion, priming with DNA expressing the trimeric V1V2 scaffold alters the hierarchy of humoral immune responses to V2 region epitopes, providing a method for more efficient induction and maintenance of V2-specific Env Abs associated with reduced risk of HIV infection.IMPORTANCE The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial.

A Parainfluenza Virus Vector Expressing the Respiratory Syncytial Virus (RSV) Prefusion F Protein Is More Effective than RSV for Boosting a Primary Immunization with RSV.

Live-attenuated pediatric vaccines for intranasal administration are being developed for human respiratory syncytial virus (RSV), an important worldwide pediatric respiratory pathogen that lacks a licensed vaccine or suitable antiviral drug. We evaluated a prime-boost strategy in which primary immunization with RSV was boosted by secondary immunization with RSV or with a chimeric recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3) vector expressing the RSV fusion F protein. The vector-expressed F protein had been engineered (DS-Cav1 mutations) for increased stability in the highly immunogenic prefusion (pre-F) conformation, with or without replacement of its transmembrane and cytoplasmic tail domains with their counterparts from bovine parainfluenza virus type 3 (BPIV3) F protein to direct incorporation into the vector virion for increased immunogenicity. In hamsters that received a primary infection with RSV, a booster infection with RSV ∼6 weeks later was completely restricted for producing infectious virus but induced a significant increase in the serum RSV-plaque-reduction neutralizing antibody titer (RSV-PRNT). Boosting instead with the rB/HPIV3-RSV-pre-F vectors resulted in efficient replication and induced significantly higher RSV-PRNTs than RSV. In African green monkeys that received a primary infection with RSV, a booster infection with RSV ∼2, ∼6, or ∼15 months later was highly restricted, whereas booster infections with the vectors had robust replication. Compared with RSV, boosts with the vectors induced 7- to 15-fold higher titers of RSV-specific serum antibodies with high neutralizing activity, as well as significantly higher titers of RSV-specific mucosal IgA antibodies. These findings support further development of this heterologous prime-boost strategy.IMPORTANCE Immune responses to RSV in infants can be reduced due to immunological immaturity and immunosuppression by RSV-specific maternal antibodies. In infants and young children, two infections with wild-type RSV typically are needed to achieve the titers of RSV-specific serum antibodies and protection against illness that are observed in adults. Therefore, a boost might substantially improve the performance of live pediatric RSV vaccines presently being developed. Hamsters and African green monkeys received a primary intranasal infection with RSV and were given a boost with RSV or a parainfluenza virus (PIV) vector expressing RSV fusion protein engineered for enhanced immunogenicity. The RSV boost was highly restricted but induced a significant increase in serum RSV-neutralizing antibodies. The PIV vectors replicated efficiently and induced significantly higher antibody responses. The use of an attenuated PIV vector expressing RSV antigen to boost a primary immunization with an attenuated RSV warrants further evaluation.

Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine.

BACKGROUND: Zika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat. METHODS: Here, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime-boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost. RESULTS: These data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays. CONCLUSIONS: Although BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

In vivo delivery of a multiepitope peptide and Nef protein using novel cell-penetrating peptides for development of HIV-1 vaccine candidate.

OBJECTIVES: A potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323, and P248-151 epitopes was designed using bioinformatics tools. The pcDNA3.1-nef-vpr-gp160-p24 and pcDNA3.1-nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated inE. coli. These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice. RESULTS: The recombinant DNA constructs were confirmed as the ~ 867 bp and ~ 648 bp bands related tonef-vpr-gp160-p24 andnef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the ~ 32 kDa and ~ 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-γ and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses. CONCLUSIONS: The heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.