Heterologous Prime-Boost Vaccination with Inactivated Influenza Viruses Induces More Effective Cross-Protection than Homologous Repeat Vaccination.

With concerns about the efficacy of repeat annual influenza vaccination, it is important to better understand the impact of priming vaccine immunity and develop an effective vaccination strategy. Here, we determined the impact of heterologous prime-boost vaccination on inducing broader protective immunity compared to repeat vaccination with the same antigen. The primed mice that were intramuscularly boosted with a heterologous inactivated influenza A virus (H1N1, H3N2, H5N1, H7N9, H9N2) vaccine showed increased strain-specific hemagglutination inhibition titers against prime and boost vaccine strains. Heterologous prime-boost vaccination of mice with inactivated viruses was more effective in inducing high levels of IgG antibodies specific for groups 1 and 2 hemagglutinin stalk domains, as well as cross-protection, compared to homologous vaccination. Both humoral and T cell immunity were found to play a critical role in conferring cross-protection by heterologous prime-boost vaccination. These results support a strategy to enhance cross-protective efficacy by heterologous prime-boost influenza vaccination.

Efficient stabilization of therapeutic hepatitis B vaccine components by amino-acid formulation maintains its potential to break immune tolerance.

Background & Aims: Induction of potent, HBV-specific immune responses is crucial to control and finally cure HBV. The therapeutic hepatitis B vaccine TherVacB combines protein priming with a Modified Vaccinia virus Ankara (MVA)-vector boost to break immune tolerance in chronic HBV infection. Particulate protein and vector vaccine components, however, require a constant cooling chain for storage and transport, posing logistic and financial challenges to vaccine applications. We aimed to identify an optimal formulation to maintain stability and immunogenicity of the protein and vector components of the vaccine using a systematic approach. Methods: We used stabilizing amino acid (SAA)-based formulations to stabilize HBsAg and HBV core particles (HBcAg), and the MVA-vector. We then investigated the effect of lyophilization and short- and long-term high-temperature storage on their integrity. Immunogenicity and safety of the formulated vaccine was validated in HBV-naïve and adeno-associated virus (AAV)-HBV-infected mice. Results: In vitro analysis proved the vaccine's stability against thermal stress during lyophilization and the long-term stability of SAA-formulated HBsAg, HBcAg and MVA during thermal stress at 40 °C for 3 months and at 25 °C for 12 months. Vaccination of HBV-naïve and AAV-HBV-infected mice demonstrated that the stabilized vaccine was well tolerated and able to brake immune tolerance established in AAV-HBV mice as efficiently as vaccine components constantly stored at 4 °C/-80 °C. Even after long-term exposure to elevated temperatures, stabilized TherVacB induced high titre HBV-specific antibodies and strong CD8+ T-cell responses, resulting in anti-HBs seroconversion and strong suppression of the virus in HBV-replicating mice. Conclusion: SAA-formulation resulted in highly functional and thermostable HBsAg, HBcAg and MVA vaccine components. This will facilitate global vaccine application without the need for cooling chains and is important for the development of prophylactic as well as therapeutic vaccines supporting vaccination campaigns worldwide. Impact and implications: Therapeutic vaccination is a promising therapeutic option for chronic hepatitis B that may enable its cure. However, its application requires functional cooling chains during transport and storage that can hardly be guaranteed in many countries with high demand. In this study, the authors developed thermostable vaccine components that are well tolerated and that induce immune responses and control the virus in preclinical mouse models, even after long-term exposure to high surrounding temperatures. This will lower costs and ease application of a therapeutic vaccine and thus be beneficial for the many people affected by hepatitis B around the world.

Heterologous prime boost COVID 19 vaccination.

Heterologous prime boost vaccination is a primary vaccination with different vaccines, most often from different vaccine platforms. It combines the immunological properties of the different vaccines and thereby induces humoral, cellular and, in some cases, mucosal response. For Covid prevention, it has been used in primary vaccination, due to safety issues and in boosters. We have evaluated some articles reporting on the results of this type of vaccine, and demonstrating its usefulness.

Antibody and T Cell Responses against SARS-CoV-2 Elicited by the Third Dose of BBIBP-CorV (Sinopharm) and BNT162b2 (Pfizer-BioNTech) Vaccines Using a Homologous or Heterologous Booster Vaccination Strategy.

In the present study, antibody and T cell-mediated immune responses elicited by BBIBP-CorV and BNT162b2 vaccines were compared 6 months after the two-dose immunization of healthy individuals. Additionally, antibody and T cell responses after the third dose of BBIBP-CorV or BNT162b2 were compared using a homologous or heterologous vaccination strategy. The third dose was consistently administered 6 months after the second dose. Six months following the two-dose vaccination, the cumulative IFNγ-positive T cell response was almost identical in participants immunized with either two doses of BNT162b2 or BBIBP-CorV vaccines; however, significant differences were revealed regarding humoral immunity: the two-dose BNT162b2 vaccine maintained a significantly higher antireceptor-binding domain (RBD) IgG, anti-spike (S1/S2) IgG, and IgA antibody levels. The BNT162b2 + BNT162b2 + BBIBP-CorV vaccine series elicited significantly lower anti-RBD IgG and anti-S1/S2 IgG levels than three doses of BNT162b2, while the anti-S IgA level was equally negligible in both groups. Importantly, the cumulative IFNγ-positive T cell response was highly similar in both groups. Surprisingly, the BBIBP-CorV + BBIBP-CorV + BNT162b2 vaccination series provided a much higher cumulative IFNγ-positive T cell response than that elicited by three doses of BNT162b2; moreover, the levels of anti-RBD IgG and anti-S IgA were almost identical. Only the mean anti-S1/S2 IgG levels were higher after receiving three mRNA vaccines. Based on these data, we can conclude that administering a third dose of BNT162b2 after two doses of BBIBP-CorV is an effective strategy to significantly enhance both humoral and T cell-mediated immune response, and its effectiveness is comparable to that of three BNT162b2 vaccines.

Higher SARS-CoV-2 Spike Binding Antibody Levels and Neutralization Capacity 6 Months after Heterologous Vaccination with AZD1222 and BNT162b2.

Within a year after the emergence of SARS-CoV-2, several vaccines had been developed, clinically evaluated, proven to be efficacious in preventing symptomatic disease, and licensed for global use. The remaining questions about the vaccines concern the duration of protection offered by vaccination and its efficacy against variants of concern. Therefore, we set out to analyze the humoral and cellular immune responses 6 months into homologous and heterologous prime-boost vaccinations. We recruited 190 health care workers and measured their anti-spike IgG levels, their neutralizing capacities against the Wuhan-Hu-1 strain and the Delta variant using a surrogate viral neutralization test, and their IFNγ-responses towards SARS-CoV-2-derived spike peptides. We here show that IFNγ secretion in response to peptide stimulation was significantly enhanced in all three vaccination groups and comparable in magnitude. In contrast, the heterologous prime-boost regimen using AZD1222 and BNT162b2 yielded the highest anti-spike IgG levels, which were 3-4.5 times more than the levels resulting from homologous AZD1222 and BNT162b2 vaccination, respectively. Likewise, the neutralizing capacity against both the wild type as well as the Delta receptor binding domains was significantly higher following the heterologous prime-boost regimen. In conclusion, our results suggest that mixing different SARS-CoV-2 vaccines might lead to more efficacious and longer-lasting humoral protection against breakthrough infections.

Current perspectives for SARS-CoV-2 vaccination efficacy improvement in patients with active treatment against cancer.

A higher risk of death from coronavirus disease 19 has been shown for patients with solid cancers or haematological malignancies (HM). Thanks to the accelerated development of anti-SARS-SoV-2 vaccines in less than a year since the start of the global pandemic, patients with cancer were quickly prioritised in early 2021 for vaccination, however dependent on the very unequal availability at the global level. Impaired immunogenicity of SARS-CoV-2 mRNA vaccines in immunocompromised patients was rapidly reported as early as April 2021, although the vaccination fortunately appears to be generally effective without increasing the spacing. Worryingly, the humoral response of the SARS-CoV-2 vaccination is, however, considered insufficient in patients followed for HM, in particular when they are on anti-CD20 treatment. Thus, improving vaccination coverage by strengthening immune stimulation should be evaluated in patients under active treatment against cancer. Here, we discuss three different approaches: a third dose of early vaccine (repeated immune stimulation), heterologous prime-boost vaccination (multimodal immune stimulation) and a double-dose strategy (maximisation of immune response). Dedicated therapeutic trials, currently almost non-existent, seem rapidly necessary.

Exploring heterologous prime-boost vaccination approaches to enhance influenza control in pigs.

Influenza A viruses evolve rapidly to escape host immunity. In swine, this viral evolution has resulted in the emergence of multiple H1 and H3 influenza A virus (IAV) lineages in the United States (US) pig populations. The heterologous prime-boost vaccination strategy is a promising way to deal with diverse IAV infection in multiple animal models. However, whether or not this vaccination strategy is applicable to US swine to impart immunity against infection from North American strains of IAV is still unknown. We performed a vaccination-challenge study to evaluate the protective efficacy of using multivalent inactivated vaccine and/or a live attenuated IAV vaccine (LAIV) in pigs following multiple prime-boost vaccination protocols against a simultaneous H1N1 and H3N2 IAV infection. Our data show that pigs in the heterologous prime-boost vaccination group had more favorable outcomes consistent with a better response against virus challenge than non-vaccinated pigs. Additionally, delivering a multivalent heterologous inactivated vaccine boost to pigs following a single LAIV administration was also beneficial. We concluded the heterologous prime boost vaccination strategy may potentiate responses to suboptimal immunogens and holds the potential applicability to control IAV in the North American swine industry. However, more studies are needed to validate the application of this vaccination approach under field conditions.

Comparative analysis of two HIV-1 multiepitope polypeptides for stimulation of immune responses in BALB/c mice.

A licensed vaccine against human immunodeficiency virus-1 (HIV-1) infection has not become available up to now. Hence, it is more rational to use immune-informatics tools for prediction of T cell epitopes (in silico study) and development of an effective epitope-driven vaccine against hypervariable pathogens. Multiepitope vaccines were considered as the next generation of an effective vaccine against HIV-1 infection. In the current study, we developed two different constructs encoding T cell epitopes derived from Nef, Vif, Vpu, Gp160 and P24 proteins in BALB/c mice. To overcome their poor immunogenicity, four different cell penetrating peptides (MPG and HR9 for DNA delivery, and CyLoP-1 and LDP-NLS for protein delivery), Montanide adjuvant, and heterologous prime-boost immunization strategy were utilized. The generation of cytokines, Granzyme B, and total IgG and its subclasses was determined using ELISA. Our data indicated that the levels of IFN-γ and Granzyme B in mice injected with Nef-Vif-Gp160-P24 multiepitope constructs were higher than those immunized with Nef-Vpu-Gp160-P24 multiepitope constructs. Moreover, the heterologous DNA priming/ multiepitope peptide boosting in both Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 regimens induced significantly high antigen-specific IgG2a and IgG2b responses in comparison with other groups. There was no significant difference between MPG and HR9 as well as CyLoP-1 and LDP-NLS as a delivery system for enhancement of immune responses. Generally, the heterologous DNA prime/ multiepitope peptide boost modalities for both constructs could significantly enhance the levels of IgG2a, IgG2b, IFN-γ, and Granzyme B directed toward Th1 immune responses as compared to homologous prime/ boost with DNA or polypeptide constructs.

CpG enhances the immunogenicity of heterologous DNA-prime/protein-boost vaccination with the heavy chain myosin of Brugia malayi in BALB/c mice.

The recombinant heavy chain myosin of Brugia malayi (Bm-Myo) has earlier been reported as a potent vaccine candidate in our lab. Subsequently, we further enhanced its efficacy employing heterologous DNA prime/protein boost (Myo-pcD+Bm-Myo) immunization approach that produced superior immune-protection than protein or DNA vaccination. In the present study, we evaluated the efficacy of heterologous prime boost vaccination in combination with CpG, synthetic oligodeoxynucleotides (ODN) adjuvant in BALB/c mice. The results showed that CpG/Myo-pcD+Bm-Myo conferred 84.5 ± 0.62% protection against B. malayi infective larval challenge which was considerably higher than Myo-pcD+Bm-Myo (75.6 ± 1.10%) following immunization. Although, both the formulations of immunization elicited robust production of specific IgG antibody and their isotypes (IgG1, IgG2a, IgG2b, and IgG3); however, CpG/Myo-pcD+Bm-Myo predominantly enhanced the level of IgG2a suggesting Th1 biased immune response in presence of CpG. Furthermore, spleen isolated from mice that immunized with CpG/Myo-pcD+Bm-Myo had greater accumulation of CD4+, CD8+, and CD19+ B cells and there was an augmented expression of co-stimulatory molecules CD40, CD86 on host dendritic cells (DCs). In contrast to Myo-pcD+Bm-Myo group, the splenocytes of CpG/Myo-pcD+Bm-Myo immunized mice developed comparatively higher pro-inflammatory cytokines IL-2 and IFN-γ leaving anti-inflammatory cytokine levels unchanged. Moreover, CpG formulation also upregulated the RNA expression of IL-12 and TNF-α in spleenocytes. The current findings suggest that the use of CpG would be more advantageous as an adjuvant predominantly in DNA/protein prime boost vaccine against Bm-Myo and presumably also for filarial infection.

Transcriptional response to a prime/boost vaccination of chickens with three vaccine variants based on HA DNA and Pichia-produced HA protein.

Highly pathogenic avian influenza causes severe economic losses and is a potential threat to public health. Better knowledge of the mechanisms of chicken response to the novel types of vaccines against avian influenza might be helpful in their successful implementation into poultry vaccination programs in different countries. This work presents a comprehensive analysis of gene expression response elicited in chicken spleens by a combined DNA/recombinant protein prime/boost vaccination compared to DNA/DNA and protein/protein regimens. All groups of vaccinated chickens displayed changes in spleen transcriptomes in comparison to the control group with 423, 375 and 212 identified differentially expressed genes in protein/protein, DNA/DNA and DNA/protein group, respectively. Genes with most significantly changed expression belong to immune-related categories. Depending on a group, a fraction of 15-34% of up-regulated and a fraction of 15-42% of down-regulated immune-related genes are shared by all groups. Interestingly, the most upregulated genes encode ß-defensins, short peptides with antimicrobial activity and immunomodulatory functions. Microarray results were validated with RT-qPCR method, which confirmed differential regulation of the selected immune-related genes. Immune-related differentially expressed genes and metabolic pathways identified in this work are compared to the available literature data on gene expression changes in vaccinated and non-vaccinated chickens after influenza infection.

Listeria-Vectored Vaccine Expressing the Mycobacterium tuberculosis 30-Kilodalton Major Secretory Protein via the Constitutively Active prfA* Regulon Boosts Mycobacterium bovis BCG Efficacy against Tuberculosis.

A potent vaccine against tuberculosis, one of the world's deadliest diseases, is needed to enhance the immunity of people worldwide, most of whom have been vaccinated with the partially effective Mycobacterium bovis BCG vaccine. Here we investigate novel live attenuated recombinant Listeria monocytogenes (rLm) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein (r30/antigen 85B [Ag85B]) (rLm30) as heterologous booster vaccines in animals primed with BCG. Using three attenuated L. monocytogenes vectors, L. monocytogenes ΔactA (LmI), L. monocytogenes ΔactA ΔinlB (LmII), and L. monocytogenes ΔactA ΔinlB prfA* (LmIII), we constructed five rLm30 vaccine candidates expressing r30 linked in frame to the L. monocytogenes listeriolysin O signal sequence and driven by the hly promoter (h30) or linked in frame to the ActA N-terminal 100 amino acids and driven by the actA promoter (a30). All five rLm30 vaccines secreted r30 in broth and macrophages; while rLm30 expressing r30 via a constitutively active prfA* regulon (rLmIII/a30) expressed the largest amount of r30 in broth culture, all five rLm30 vaccines expressed equivalent amounts of r30 in infected macrophages. In comparative studies, boosting of BCG-immunized mice with rLmIII/a30 induced the strongest antigen-specific T-cell responses, including splenic and lung polyfunctional CD4+ T cells expressing the three cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) (P < 0.001) and splenic and lung CD8+ T cells expressing IFN-γ (P < 0.0001). In mice and guinea pigs, the rLmIII/a30 and rLmI/h30 vaccines were generally more potent booster vaccines than r30 with an adjuvant and a recombinant adenovirus vaccine expressing r30. In a setting in which BCG alone was highly immunoprotective, boosting of mice with rLmIII/a30, the most potent of the vaccines, significantly enhanced protection against aerosolized M. tuberculosis (P < 0.01).

In vivo electroporation in DNA-VLP prime-boost preferentially enhances HIV-1 envelope-specific IgG2a, neutralizing antibody and CD8 T cell responses.

Although in vivo electroporation (EP) has been utilized to improve immunogenicity in DNA vaccines alone or in prime-boost regimens with both proteins and viral-vectors, no studies on in vivo EP in DNA-VLP prime-boost regimens against HIV-1 have been reported. Previously we developed stably transfected Drosophila S2 clones to produce HIV-1 virus-like particles (VLP) and demonstrated that priming mice twice with DNA plasmids encoding HIV-1 gp120 and gag and boosting twice with HIV-1 VLP (i.e. DDVV immunization) elicited both envelope-specific antibody and envelope and gag-specific CD8 T cell responses. However, the potency and the breadth of immunogenicity still need to be improved. In this study we tested the effect of in vivo EP during DNA priming on immunogenicity of this DNA-VLP prime-boost regimen. Here we report that although both DDVV and DDVV+EP elicited gp120-specific ELISA-binding antibody responses, average EC50 values of gp120-specific ELISA-binding total IgG, IgG2a, but not IgG1, antibody responses were significantly higher in DDVV+EP than in DDVV. Moreover, while DDVV elicited neutralizing antibody responses against autologous, but not other five, strains tested, DDVV+EP not only elicited significantly higher anti-autologous neutralizing antibody responses, but also cross-neutralizes four of five other HIV-1 strains tested, including two tier 2 strains. Finally, although CD4 and CD8 T cells from both DDVV and DDVV+EP immunizations secreted IFN-γ, IL-2, TNF-α upon HIV-1 envelope peptide stimulation, average HIV-1 envelope-specific CD8 T cells that secreted IFN-γ, IL-2 and/or TNF-α were significantly higher in DDVV+EP than in DDVV. Thus we conclude that DDVV+EP immunization preferentially increases HIV-1 envelope-specific TH1 cytokine-mediated IgG2a responses and significantly enhances the potency and the breadth of neutralizing antibody responses including tier 2 viruses. Further study on this heterologous regimen in large animals is warranted.

An optimized peptide vaccine strategy capable of inducing multivalent CD8+ T cell responses with potent antitumor effects.

Therapeutic cancer vaccines are an attractive alternative to conventional therapies for treating malignant tumors, and successful tumor eradication depends primarily on obtaining high numbers of long-lasting tumor-reactive CD8+ T cells. Dendritic cell (DC)-based vaccines constitute a promising approach for treating cancer, but in most instances low immune responses and suboptimal therapeutic effects are achieved indicating that further optimization is required. We describe here a novel vaccination strategy with peptide-loaded DCs followed by a mixture of synthetic peptides, polyinosine-polycytidylic acid (poly-IC) and anti-CD40 antibodies (TriVax) for improving the immunogenicity and therapeutic efficacy of DC-based vaccines in a melanoma mouse model. TriVax immunization 7-12 d after priming with antigen-loaded DCs generated large numbers of long-lasting multiple antigen-specific CD8+ T cells capable of recognizing tumor cells. These responses were far superior to those generated by homologous immunizations with either TriVax or DCs. CD8+ T cells but not CD4+ T cells or NK cells mediated the therapeutic efficacy of this heterologous prime-boost strategy. Moreover, combinations of this vaccination regimen with programmed cell death-1 (PD-1) blockade or IL2 anti-IL2 antibody complexes led to complete disease eradication and survival enhancement in melanoma-bearing mice. The overall results suggest that similar strategies would be applicable for the design of effective therapeutic vaccination for treating viral diseases and various cancers, which may circumvent current limitations of cell-based cancer vaccines.

CD8+ T Cell-mediated Immunity Induced by Heterologous Prime-boost Vaccination Based on DNA Vaccine and Recombinant Vaccinia Virus Expressing Epitope

BACKGROUND: DNA vaccination represents an anticipated approach for the control of numerous infectious diseases. Used alone, however, DNA vaccine is weak immunogen inferior to viral vectors. In recent, heterologous prime-boost vaccination leads DNA vaccines to practical reality. METHODS: We assessed prime-boost immunization strategies with a DNA vaccine (minigene, gB498-505 DNA) and recombinant vaccinia virus (vvgB498- 505) expressing epitope gB498-505 (SSIEFARL) of CD8+ T cells specific for glycoprotein B (gB) of herpes simplex virus (HSV). Animals were immunized primarily with gB498-505 epitope-expressing DNA vaccine/recombinant vaccinia virus and boosted with alternative vaccine type expressing entire Ag. RESULTS: In prime-boost protocols using vvgBw (recombinant vaccinia virus expressing entire Ag) and vvgB498-505, CD8+ T cell-mediated immunity was induced maximally at both acute and memory stages if primed with vvgBw and boosted with vvgB498-505 as evaluated by CTL activity, intracellular IFN-staining, and MHC class I tetramer staining. Similarly gB498-505 DNA prime-gBw DNA (DNA vaccine expressing entire Ag) boost immunization elicited the strongest CD8+ T cell responses in protocols based on DNA vaccine. However, the level of CD8+ T cell-mediated immunity induced with prime-boost vaccination using DNA vaccine expressing epitope or entire Ag was inferior to those based on vvgBw and vvgB498-505. Of particular interest CD8+ T cell-mediated immunity was optimally induced when vvgB498-505 was used to prime and gB DNA was used as alternative boost. Especially CD8+ T cell responses induced by such protocol was longer lasted than other protocols. CONCLUSION: These facts direct to search for the effective strategy to induce optimal CD8+ T cell-mediated immunity against cancer and viral infection.

Influence of Immunity Induced at Priming Step on Mucosal Immunization of Heterologous Prime-Boost Regimens

BACKGROUND: The usefulness of DNA vaccine at priming step of heterologous prime-boost vaccination led to DNA vaccine closer to practical reality. DNA vaccine priming followed by recombinant viral vector boosting via systemic route induces optimal systemic immunity but no mucosal immunity. Mucosal vaccination of the reversed protocol (recombinant viral vector priming-DNA vaccine boosting), however, can induce both maximal mucosal and systemic immunity. Here, we tried to address the reason why the mucosal protocol of prime-boost vaccination differs from that of systemic vaccination. METHODS: To address the importance of primary immunity induced at priming step, mice were primed with different doses of DNA vaccine or coadministration of DNA vaccine plus mucosal adjuvant, and immunity including serum IgG and mucosal IgA was then determined following boosting with recombinant viral vector. Next, to assess influence of humoral pre-existing immunity on boosting CD8+ T cell-mediated immunity, CD8+ T cell-mediated immunity in B cell-deficient (microK/O) mice immunized with prime-boost regimens was evaluated by CTL assay and IFN-gamma-producing cells. RESULTS: Immunity primed with recombinant viral vector was effectively boosted with DNA vaccine even 60 days later. In particular, animals primed by increasing doses of DNA vaccine or incorporating an adjuvant at priming step and boosted by recombinant viral vector elicited comparable responses to recombinant viral vector primed-DNA vaccine boosted group. Humoral pre-existing immunity was also unlikely to interfere the boosting effect of CD8+ T cell-mediated immunity by recombinant viral vector. CONCLUSION: This report provides the important point that optimally primed responses should be considered in mucosal immunization of heterologous prime-boost regimens for inducing the effective boosting at both mucosal and systemic sites.