A multistage-polyepitope vaccine protects against Mycobacterium tuberculosis infection in HLA-DR3 transgenic mice

Mycobacterium tuberculosis (Mtb) is responsible for almost 2 million deaths annually. BCG, currently the only TB vaccine, induces variable protection and does not protect against reactivation of latent TB. Thus, efficient vaccines to supplement BCG are required urgently. Since Mtb's proteome differs qualitatively and quantitatively during bacterial replication stages from that expressed during dormancy, improved TB vaccines should drive immune responses to Mtb antigens expressed during multiple stages of infection. Consequently, such “multistage” vaccines should be composed of (immunodominant) antigens expressed during different phases of Mtb infection. As a concept multistage vaccine, we constructed a polyepitope by fusing five HLA-DR3-restricted T-cell epitopes derived from different Mtb proteins either expressed highly by replicating bacteria (Ag85B, hsp65, 19 kDa lipoprotein), or abundantly expressed by dormant bacilli and recognized preferentially by TST⁺ individuals (hsp16, Rv1733c). PBMC of HLA-DR3⁺ but not HLA-DR3⁻ cured TB patients and TST⁺ individuals responded well to the multistage-polyepitope in vitro. The in vivo immunogenicity and protective efficacy of the multistage-polyepitope were analyzed using HLA-DR3 transgenic mice lacking endogenous murine class II as a model. Immunization with the multistage-polyepitope adjuvanted with CpG generated high IgG levels as well as polyfunctional CD4⁺ T-cells producing IFN-γ, TNF and IL-2, specific for these HLA-DR3-restricted epitopes. Importantly, multistage-polyepitope immunization reduced the number of bacilli in the lungs after Mtb challenge when administered as prophylactic vaccine. Given the extensive repertoire of potential Mtb antigens available for immune recognition, the data of our model demonstrate the potential of multistage-polyepitope vaccines to protect against TB.     

Universal influenza DNA vaccine encoding conserved CD4 T cell epitopes protects against lethal viral challenge in HLA-DR transgenic mice

The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid™ in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4⁺ T cell responses to protection against influenza infection.     

Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB110401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4⁺ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50–70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50 days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9 dpc. While contraction rates of Th cells and the inflammatory response during 14–35 dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9–14 days. The FAD group approached moribund by 14 dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.     

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI).Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine.This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus.     

A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax

The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10⁴ were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10⁵. All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in highlighting the efficacy of plant expressed PA(dIV) by oral immunization in murine model.     

A novel HSV-2 subunit vaccine induces GLA-dependent CD4 and CD8 T cell responses and protective immunity in mice and guinea pigs

Background/objectivesThere is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection.MethodsWe developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination.ResultsVaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score.ConclusionsTaken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.     

Therapeutic efficacy of the multi-epitope vaccine CTB-UE against Helicobacter pylori infection in a Mongolian gerbil model and its microRNA-155-associated immuno-protective mechanism

Vaccination is an effective means of preventing infectious diseases, including those caused by Helicobacter pylori. In this study, we constructed a novel multi-epitope vaccine, CTB-UE, composed of the cholera toxin B subunit and tandem copies of the B and Th cell epitopes from the H. pylori urease A and B subunits. We evaluated the therapeutic efficacy of the multi-epitope vaccine CTB-UE against H. pylori infection in a Mongolian gerbil model and studied its immuno-protective mechanisms. The experimental results indicated that urease activity, H. pylori colonisation density, the levels of IL-8 and TNF-α in the serum, and the levels of COX-2 and NAP in gastric tissue were significantly lower and the IgG level in the serum and the IFN-γ level in spleen lymphocytes were significantly higher in the vaccinated group compared with the model control group; additionally, gastric mucosal inflammation was notably alleviated following vaccination. The results showed that CTB-UE had a good therapeutic effect on H. pylori infection. The immuno-protective mechanism was closely related to the immune response mediated by microRNA-155, the expression of which was strongly up-regulated after CTB-UE administration. The expression levels of the microRNA-155 target proteins IFN-γRα, AID, and PU.1 were significantly down-regulated; these results indicated that CTB-UE induced an immune response biased towards Th1 cells by up-regulating microRNA-155 to inhibit IFN-γRα expression and induced a humoral immune response towards B cells by up-regulating microRNA-155 to inhibit PU.1 and AID expression. These results demonstrate that the multi-epitope vaccine CTB-UE may be a promising therapeutic vaccine against H. pylori infection and is a new therapeutic tool for human use.     

Differential effect of CD4Foxp3 T-regulatory cells on the B and T helper cell responses to influenza virus vaccination

The T-regulatory (T-reg) cells restrict the T-cell functions in various viral infections including influenza infection. However little is known about the effect of T-regs in influenza vaccination. Herein, we found that immunization of BALB/c mice with a prototype of UV-inactivated influenza PR8/A/34 virus vaccine expanded the CD4⁺Foxp3⁺ T-reg pool and fostered the development of virus-specific CD4⁺Foxp3⁺ T-reg cells. Increasing the size of Foxp3⁺ T-reg pool did not alter the primary PR8-specific B-cell response, but it did suppress the primary and memory PR8-specific T helper responses induced by vaccination. In contrast, the vaccination-induced T helper cell response was augmented in the absence of CD4⁺Foxp3⁺ T-reg cells. Since CD4 T helper cells contribute to anti-influenza protection, therapeutic “quenching” of T-reg function prior to vaccination may enhance the efficacy of influenza vaccination.     

Differential activation of host cell signalling pathways through infection with two variants of influenza A/Puerto Rico/8/34 (H1N1) in MDCK cells

In cell culture-based influenza vaccine production, few efforts have been undertaken to characterise virus–host cell interactions in detail. Two influenza virus strains that grew to different virus titres, and differed in virus dynamics, apoptosis induction and proteome changes were observed.     

A novel nanoemulsion vaccine induces mucosal Interleukin-17 responses and confers protection upon Mycobacterium tuberculosis challenge in mice

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is contracted via aerosol infection, typically affecting the lungs. Mycobacterium bovis bacillus Calmette-Guerin (BCG) is the only licensed vaccine and has variable efficacy in protecting against pulmonary TB. Additionally, chemotherapy is associated with low compliance contributing to development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Thus, there is an urgent need for the design of more effective vaccines against TB. Experimental vaccines delivered through the mucosal route induce robust T helper type 17 (Th17)/ Interleukin (IL) -17 responses and provide superior protection against Mtb infection. Thus, the development of safe mucosal adjuvants for human use is critical. In this study, we demonstrate that nanoemulsion (NE)-based adjuvants when delivered intranasally along with Mtb specific immunodominant antigens (NE-TB vaccine) induce potent mucosal IL-17 T-cell responses. Additionally, the NE-TB vaccine confers significant protection against Mtb infection, and when delivered along with BCG, is associated with decreased disease severity. These findings strongly support the development of a NE-TB vaccine as a novel, safe and effective, first-of-kind IL-17 inducing mucosal vaccine for potential use in humans.     

Covax-19/Spikogen® vaccine based on recombinant spike protein extracellular domain with Advax-CpG55.2 adjuvant provides single dose protection against SARS-CoV-2 infection in hamsters

COVID-19 presents an ongoing global health crisis. Protein-based COVID-19 vaccines that are well-tolerated, safe, highly-protective and convenient to manufacture remain of major interest. We therefore sought to compare the immunogenicity and protective efficacy of a number of recombinant SARS-CoV-2 spike protein candidates expressed in insect cells. By comparison to a full length (FL) spike protein detergent-extracted nanoparticle antigen, the soluble secreted spike protein extracellular domain (ECD) generated higher protein yields per liter of culture and when formulated with either Alum-CpG55.2 or Advax-CpG55.2 combination adjuvants elicited robust antigen-specific humoral and cellular immunity in mice. In hamsters, the spike ECD when formulated with either adjuvant induced high serum neutralizing antibody titers even after a single dose. When challenged with the homologous SARS-CoV-2 virus, hamsters immunized with the adjuvanted spike ECD exhibited reduced viral load in day 1–3 oropharyngeal swabs and day 3 nasal turbinate tissue and had no recoverable infectious virus in day 3 lung tissue. The reduction in lung viral load correlated with less weight loss and lower lung pathology scores. The formulations of spike ECD with Alum-CpG55.2 or Advax-CpG55.2 were protective even after just a single dose, although the 2-dose regimen performed better overall and required only half the total amount of antigen. Pre-challenge serum neutralizing antibody levels showed a strong correlation with lung protection, with a weaker correlation seen with nasal or oropharyngeal protection. This suggests that serum neutralizing antibody levels may correlate more closely with systemic, rather than mucosal, protection. The spike protein ECD with Advax-CpG55.2 formulation (Covax-19® vaccine) was selected for human clinical development.     

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays,antigen-specific antibody assays and cellular assays

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax?), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M?052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.