Immunogenicity of RepliVAX WN, a novel single-cycle West Nile virus vaccine

We recently reported that immunization with RepliVAX WN, a single-cycle West Nile virus (WNV) vaccine, protected mice against WNV challenge. We have extended these studies by characterizing the RepliVAX WN-elicited antibody and T cell responses. WNV-specific IgG antibody responses comprised predominantly of IgG_2c and IgG_2b subclasses were detected 8 months after immunization. Vigorous WNV-specific CD4⁺ and CD8⁺ T cell responses directed at both structural and nonstructural WNV proteins were detected which were characterized by cytolytic activity and secretion of IFN-γ and TNF-α. Importantly, RepliVAX WN immunization resulted in vigorous CD8⁺ memory T cell responses detected at 8 months after immunization.     

Lack of neonatal Fc receptor does not diminish the efficacy of the HSV-1 0ΔNLS vaccine against ocular HSV-1 challenge

The neonatal Fc receptor (FcRn) is constitutively expressed in the cornea and is up-regulated in response to herpes simplex virus type 1 (HSV-1). Previously, we found targeting cornea FcRn expression by small interfering RNA-mediated knockdown reduced the local efficacy of HSV-1 0ΔNLS vaccinated C57BL/6 mice against ocular challenge with HSV-1. The current study was undertaken to evaluate the HSV-1 0ΔNLS vaccine efficacy in FcRn deficient (FcRn KO) mice challenged with HSV-1. Whereas there was little neutralizing antibody detected in the serum of HSV-1 0ΔNLS vaccinated FcRn KO mice, these mice exhibited the same degree of protection against ocular challenge with HSV-1 as wild type (WT) C57BL/6 mice as measured by cumulative survival, infectious virus shed or retained in tissue, and corneal pathology including opacity and neovascularization. Mock-vaccinated FcRn KO mice were found to be more sensitive to ocular HSV-1 infection compared to mock-vaccinated (WT) mice in terms of cumulative survival and virus shedding. In addition, the FcRn KO mice generated significantly fewer effector (CD3⁺CD44⁺CD62L^-) and central (CD3⁺CD44⁺CD62L⁺) memory CD8⁺ T cells compared to the WT mice 7 days post infection. Collectively, mock-vaccinated FcRn KO mice are susceptible to ocular HSV-1 infection but HSV-1 0ΔNLS vaccinated FcRn KO mice are resistant suggesting that in addition to the FcRn, other pathways are involved in mediating the protective effect of the HSV-1 0ΔNLS vaccine against subsequent HSV-1 challenge.     

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ⁺ CD4⁺ and CD8⁺ specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis.     

CD8 T cell response in HLA-A*0201 transgenic mice is elicited by epitopes from SARS-CoV S protein

Cytotoxic CD8⁺ T lymphocytes (CTLs) play an important role in antiviral immunity. Several human HLA-A*0201 restricted CTL epitopes of severe acute respiratory syndrome (SARS) spike (S) protein have been identified in HLA-A*0201 transgenic (Tg) mice, but the mechanisms and properties of immune responses are still not well understood. In this study, HLA-A*0201 Tg mice were primed intramuscularly with SARS S DNA and boosted subcutaneously with HLA-A*0201 restricted peptides. The lymphocytes from draining lymph nodes, spleens and lungs were stimulated with the cognate peptides. Three different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses during short and long periods of time after immunization. Results showed that peptide-specific CD8⁺ T cells secreted IFN-γ, TNF-α and IL-2 and expressed CD107a/b on cell surface. IFN-γ⁺CD8⁺ T cells and CD107a/b⁺CD8⁺ T cells distributed throughout the lymphoid and non-lymphoid tissues, but the frequency of peptide-specific CD8⁺ T cells was higher in lungs than in spleens and lymph nodes. The phenotype of the CD8⁺ T cells was characterized based on the expression of IFN-γ. Most of the HLA-A*0201 restricted peptide-specific CD8⁺ T cells represented a memory subset with CD45RB^high and CD62L^low. Taken together, these data demonstrate that immunization with SARS S DNA and HLA-A*0201 restricted peptides can elicit antigen-specific CD8⁺ T cell immune responses which may have a significant implication in the long-term protection. We provide novel information in cellular immune responses of SARS S antigen-specific CD8⁺ T cells, which are important in the development of vaccine against SARS-CoV infection.     

Influence of antigen exposure on the loss of long-term memory to childhood vaccines in HIV-infected patients

The role of antigen exposure and of CD4 cell deficiency in the long-term persistence of immune memory to childhood vaccines remains uncertain, particularly during HIV infection. We analyzed in vaccinated ART-treated HIV+ patients with undetectable plasma HIV and CD4 cells >250/mm³ the persistence of two memory cell pools: effector IFNγ-producing and proliferative central memory T cells against two vaccines: (i) vaccinia against the eradicated smallpox virus, and (ii) BCG against Mtb, a persistent pathogen. None of the HIV+ patients had IFNγ-effector cells against VV while the one patient with BCG-specific effector T cells had a recent history of tuberculosis. Proliferative responses were detectable but showed significantly lower frequency and intensity of VV-specific than tuberculin-specific responses, independently of the CD4 nadir. Thus, differential patterns of persistence or recovery of T cell memory pools against childhood vaccines are observed in treated HIV infection that are governed by antigen exposure.     

Evaluation of the immune response and protective effects of rhesus macaques vaccinated with biodegradable nanoparticles carrying gp120 of human immunodeficiency virus

We previously reported that biodegradable amphiphilic poly(γ-glutamic acid) nanoparticles (NPs) carrying the recombinant gp120 env protein of the human immunodeficiency virus type 1 (HIV-1) were efficiently taken up by dendritic cells, and induced strong CD8⁺ T cell responses against the gp120 in mice. To evaluate gp120-carrying NPs (gp120-NPs) as a vaccine candidate for HIV-1 infection, we vaccinated rhesus macaques with these gp120-NPs and examined the immune response and protective efficacy against a challenge inoculation of simian and human immunodeficiency chimeric virus (SHIV). We found that gp120-NP vaccination induced stronger responses for both gp120-specific cellular and humoral immunity than gp120-alone vaccination. After the challenge inoculation with SHIV, however, the peak value of viral RNA in the peripheral blood was higher in the vaccinated groups, especially the gp120-NP vaccinated group, than naive control group. Higher value of viral load was also maintained in gp120-NP vaccinated group. Furthermore, CD4⁺ T cells from the peripheral blood decreased more in the vaccinated groups than the control group. Thus, induced immune responses against gp120 enclosed in NPs were not effective for protection but, conversely enhanced the infection, although the gp120-NPs showed a stronger induction of immune responses against the vaccinated antigen in rhesus macaques. These results support the importance of determining immune correlate of protective immunity for vaccine development against HIV-1 infection.     

Adjuvants that stimulate TLR3 or NLPR3 pathways enhance the efficiency of influenza virus-like particle vaccines in aged mice

There is intense interest in the design and use of vaccine strategies against influenza to enhance protective immune responses in the elderly. To address the need for improved influenza vaccines for the aged, two inflammatory adjuvants, Imject^® alum (a stimulator of the Nod-like receptor, Nalp3) and poly I:C (a toll-like receptor type 3 ligand), were used during vaccination with novel influenza virus-like particles (VLP). Adult (4 month old) or aged (24 month old) mice were vaccinated with VLPs alone or in combination with adjuvant. VLP-vaccinated adult mice were protected from a lethal influenza virus challenge without the use of either adjuvant. In contrast, only aged mice that were vaccinated with VLPs plus adjuvant survived challenge, whereas ∼33% of the mice vaccinated with VLP only survived challenge. Mice vaccinated with adjuvant only did not survive challenge despite similar levels of activation of CD11b⁺/CD11c⁺ dendritic cells in the lungs. The protection was not associated with HAI titers or HA specific CD8⁺ T cells, since both adjuvants boosted the VLP-induced serum HAI titers and CD8⁺ responses in adult mice, but not aged mice. Influenza VLPs used in combination with two different inflammatory adjuvants during vaccination allow for the immune system to overcome the deficiency in the aged immune system to influenza virus infection.     

Phenotype of the anti-Rickettsia CD8 T cell response suggests cellular correlates of protection for the assessment of novel antigens

The obligately intracellular bacteria Rickettsia infect endothelial cells and cause systemic febrile diseases that are potentially lethal. No vaccines are currently available and current knowledge of the effective immune response is limited. Natural and experimental rickettsial infections provide strong and cross-protective cellular immunity if the infected individual survives the acute infection. Although resistance to rickettsial infections is attributed to the induction of antigen-specific T cells, particularly CD8⁺ T cells, the identification and validation of correlates of protective cellular immunity against rickettsial infections, an important step toward vaccine validation, remains a gap in this field. Here, we show that after a primary challenge with Rickettsia typhi in the C3H mouse model, the peak of anti-Rickettsia CD8⁺ T cell-mediated responses occurs 7 days post-infection (dpi), which coincides with the beginning of rickettsial clearance. At this time point, both effector-type and memory-type CD8⁺ T cells are present, suggesting that 7 dpi is a valid time point for the assessment of CD8⁺ T cell responses of mice previously immunized with protective antigens. Based on our results, we suggest four correlates of cellular protection for the assessment of protective rickettsial antigens: (1) production of IFN-γ by antigen-experienced CD3⁺CD8⁺CD44^high cells, (2) production of Granzyme B by CD27^lowCD43^low antigen-experienced CD8⁺ T cells, (3) generation of memory-type CD8⁺ T cells [Memory Precursor Effector Cells (MPECs), as well as CD127^highCD43^low, and CD27^highCD43^low CD8⁺ T cells], and (4) generation of effector-like memory CD8⁺ T cells (CD27^lowCD43^low). We propose that these correlates could be useful for the general assessment of the quality of the CD8⁺ T cell immune response induced by novel antigens with potential use in a vaccine against Rickettsia.     

Polyanhydride nanovaccine against swine influenza virus in pigs

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200 nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4⁺CD8αα⁺ T helper and CD8⁺ cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity.     

Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime–protein boost regimen against bovine viral diarrhea virus

The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime–protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4⁺IFN-γ⁺ T cells and CD8⁺IFN-γ⁺ T cells among total CD3⁺ T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime–protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime–protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime–protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.     

Local innate and adaptive immune responses regulate inflammatory cell influx into the lungs after vaccination with formalin inactivated RSV

Inactivated respiratory syncytial virus (RSV) vaccines tend to predispose for immune mediated enhanced disease, characterized by Th2 responses and airway hypersensitivity reactions. We show in a C57BL/6 mouse model that the early innate response elicited by the challenge virus (RSV versus influenza virus) influences the outcome of the Th1/Th2 balance in the lung after intramuscular priming with inactivated vaccine. Priming of CD4⁺/IFN-γ⁺ T cells by mature dendritic cells administered intravenously and/or priming of a virus specific CD8⁺ T cell response ameliorated the Th2-mediated inflammatory response in the lung, suggesting that vaccination procedures are feasible that prevent vaccine induced immune pathology.     

Recombinant pro-apoptotic Mycobacterium tuberculosis generates CD8 T cell responses against human immunodeficiency virus type 1 Env and M. tuberculosis in neonatal mice

Mycobacterium bovis BCG is an attractive vaccine vector against breast milk HIV transmission because it elicits Th1-type responses in newborns. However, BCG causes disease in HIV-infected infants. Genetically attenuated Mycobacterium tuberculosis (Mtb) mutants represent a safer alternative for immunocompromised populations. In the current study, we compared the immunogenicity in mice of three different recombinant attenuated Mtb strains expressing an HIV envelope (Env) antigen construct. Two of these strains (ΔlysA ΔpanCD Mtb and ΔRD1 ΔpanCD Mtb) failed to induce significant levels of HIV Env-specific CD8⁺ T cell responses. In striking contrast, an HIV-1 Env-expressing attenuated ΔlysA Mtb containing a deletion in secA2, which encodes a virulence-related secretion system involved in evading adaptive immunity, generated consistently measurable Env-specific CD8⁺ T cell responses that were significantly greater than those observed after immunization with BCG expressing HIV Env. Similarly, another strain of ΔlysA ΔsecA2 Mtb expressing SIV Gag induced Gag- and Mtb-specific CD8⁺ T cells producing perforin or IFNγ, and Gag-specific CD4⁺ T cells producing IFNγ within 3 weeks after immunization in adult mice; in addition, IFNγ-producing Gag-specific CD8⁺ T cells and Mtb-specific CD4⁺ T cells were observed in neonatal mice within 1 week of immunization. We conclude that ΔlysA ΔsecA2 Mtb is a promising vaccine platform to construct a safe combination HIV-TB vaccine for use in neonates.     

Matrix-M™ adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus and an emerging pathogen in many parts of the world. In the elderly and immunosuppressed, infection can progress rapidly to debilitating and sometimes fatal neuroinvasive disease. Currently, no WNV vaccine is approved for use in humans. As there have been several recent outbreaks in the United States and Europe, there is an increasing need for a human WNV vaccine. In this study, we formulated the ectodomain of a recombinant WNV envelope (E) protein with the particulate saponin-based adjuvant Matrix-M™ and studied the antigen-specific immune responses in mice. Animals immunized with Matrix-M™ formulated E protein developed higher serum IgG1 and IgG2a and neutralizing antibody titers at antigen doses ranging from 0.5 to 10 μg compared to those immunized with 3 or 10 μg of E alone, E adjuvanted with 1% Alum, or with the inactivated virion veterinary vaccine, Duvaxyn^® WNV. This phenotype was accompanied by strong cellular recall responses as splenocytes from mice immunized with Matrix-M™ formulated vaccine produced high levels of Th1 and Th2 cytokines. Addition of Matrix-M™ prolonged the duration of the immune response, as elevated humoral and cellular responses were maintained for more than 200 days. Importantly, mice vaccinated with Matrix-M™ formulated E protein were protected from lethal challenge with both lineage 1 and 2 WNV strains. In summary, Matrix-M™ adjuvanted E protein elicited potent and durable immune responses that prevented lethal WNV infection, and thus is a promising vaccine candidate for humans.     

Intranasal delivery of whole cell lysate of Mycobacterium tuberculosis induces protective immune responses to a modified live porcine reproductive and respiratory syndrome virus vaccine in pigs

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease to pork producers worldwide. Commercially, both live and killed PRRSV vaccines are available to control PRRS, but they are not always successful. Based on the results of mucosal immunization studies in other viral models, a good mucosal vaccine may be an effective way to elicit protective immunity to control PRRS outbreaks. In the present study, mucosal adjuvanticity of Mycobacterium tuberculosis whole cell lysate (Mtb WCL) was evaluated in pigs administered a modified live PRRS virus vaccine (PRRS-MLV) intranasally. A Mtb WCL mediated increase in the frequency of NK cells, CD8⁺and CD4⁺ T cells, and γδ T cells in pig lungs were detected. Importantly, an increased and early generation of PRRSV specific neutralizing antibodies were detected in PRRS-MLV+ Mtb WCL compared to pigs inoculated with vaccine alone. In addition, there was an increased secretion of Th1 cytokines (IFNγ and IL-12) that correlated with a reciprocal reduction in the production of immunosuppressive cytokines (IL-10 and TGFβ) as well as T-regulatory cells in pigs vaccinated with PRRS-MLV+ Mtb WCL. Further, a complete rescue in arginase levels in the lungs mediated through Mtb WCL was observed in pigs inoculated with PRRS-MLV. In conclusion, Mtb WCL may be a potent mucosal adjuvant for PRRS-MLV in order to potentiate the anti-PRRSV specific immune responses to control PRRS effectively.     

Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge

The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4⁺ T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4⁺ CD44^high CD62L^low T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.     

Comparing human T cell and NK cell responses in viral-based malaria vaccine trials

Vaccination with viral-based vaccines continues to hold promise for the prevention of malaria. Whilst antigen-specific T cell responses are considered a major aim of such an approach, a role for induced NK cells as anti-malarial effector cells, or in shaping T cell responses, has received less attention. In this study naïve human volunteers were vaccinated in a prime-boost vaccination regimen comprising recombinant viral vectors fowlpox (FP9) and modified vaccinia Ankara (MVA) encoding liver-stage antigens, or a virosome vaccine. Significant T cell responses specific for the vectored vaccine antigens were demonstrated by IFNγ ELISPOT and intracellular cytokine staining (ICS) for IFNγ and IL-2, the ICS being associated with increased time to parasitaemia following subsequent challenge. Numbers of CD56^bright lymphocytes increased significantly following vaccination, as did CD3⁺ CD56⁺ lymphocytes, whilst CD56^dim cells did not. No such increases were seen with the virosome vaccine. There was no significant correlation of these CD56⁺ populations with the antigen-specific T cell responses nor time to parasitaemia. To investigate pathways of immune activation that could contribute to these lymphocyte responses, viral vectors were shown in vitro to efficiently infect APCs but not lymphocytes, and stimulated inflammatory cytokines such as type I interferons. In conclusion, measuring antigen-specific T cells is more meaningful than NK cells in these vaccination regimens.     

Recombinant Erns-E2 protein vaccine formulated with MF59 and CPG-ODN promotes T cell immunity against bovine viral diarrhea virus infection

To obtain an effective vaccine candidate against bovine viral diarrhea virus (BVDV) disease which causes great economical loss in cattle industries, recombinant E^rns-E2 protein vaccine containing MF59 and CPG-ODN adjuvants was prepared and assessed in this study. The recombinant plasmid (pET32a-E^rns-E2) was constructed and transformed into BL21 (DE3) cells to produce E^rns-E2 protein. We immunized mice with the MF59–and CPG-ODN–adjuvanted recombinant E^rns-E2 protein, E2 protein, or E^rns protein, respectively. To evaluate immunogenicity and efficacy of a vaccine-adjuvant combination, mice were challenged with BVDV BJ175170 strain after immunization. All adjuvanted vaccines elicited detectable humoral and cellular immune responses, the BVDV-specific antibody titers as well as interleukin 4 (IL-4) levels in sera of mice immunized with the recombinant E^rns-E2 protein were higher than in those of mice immunized with either the recombinant E^rns or E2 protein. Besides, immunization with the E^rns-E2 vaccines induced higher percentage of CD4⁺IFN-γ⁺, CD8⁺IFN-γ⁺ T cells and CD3⁺TNF-α⁺ T cells compared with the other vaccines. More protective efficacy against BVDV infection was acquired in the mice treated with the recombinant E^rns-E2 protein, as shown by a reduction of viremia and slight pathological changes compared with both the control mice and the other vaccinated mice. Our findings suggest that the use of the recombinant E^rns-E2 protein vaccine formulated with MF59 and CPG-ODN adjuvants enhances T cell responses and viral control, which warrants the E^rns-E2 protein vaccine-adjuvant combination could be as a vaccine strategy to against BVDV.     

A West Nile virus NS4B-P38G mutant strain induces adaptive immunity via TLR7-MyD88-dependent and independent signaling pathways

Prior work shows that an attenuated West Nile virus (WNV), the nonstructural (NS)4B-P38G mutant infection in mice induced strong immune responses and protected host from subsequent lethal wild-type WNV infection. Here, we investigated NS4B-P38G mutant infection in myeloid differentiation factor 88-deficient (MyD88^−/−) and Toll-like receptor 7-deficient (TLR7^−/−) mice and found they had enhanced susceptibility compared to wild-type mice. Both groups had lower WNV-specific IgM response and reduced effector T cell functions. Dendritic cells (DCs) also exhibited a reduced maturation and impaired antigen-presenting functions compared to wild-type DCs. Moreover, infection with NS4B-P38G mutant in TLR7^−/− and MyD88^−/− mice provided full and partial protection respectively from subsequent challenge with lethal wild-type WNV. There were reduced T cell responses in MyD88^−/− and interleukin-1 receptor deficient (IL-1R^−/−) mice during secondary challenge with wild-type WNV. In contrast, TLR7^−/− mice displayed normal T cell functions. Collectively, these results suggest that TLR7-dependent MyD88 signaling is required for T cell priming during NS4B-P38G mutant infection, whereas the TLR7-independent MyD88 signaling pathways are involved in memory T cell development, which may contribute to host protection during secondary challenge with wild-type WNV     

A novel lipid nanoparticle adjuvant significantly enhances B cell and T cell responses to sub-unit vaccine antigens

Sub-unit vaccines are primarily designed to include antigens required to elicit protective immune responses and to be safer than whole-inactivated or live-attenuated vaccines. But their purity and inability to self-adjuvant often result in weaker immunogenicity. Emerging evidence suggests that bio-engineered nanoparticles can be used as immunomodulatory adjuvants. Therefore, in this study we explored the potential of novel Merck-proprietary lipid nanoparticle (LNP) formulations to enhance immune responses to sub-unit viral antigens. Immunization of BALB/c and C57BL/6 mice revealed that LNPs alone or in combination with a synthetic TLR9 agonist, immune-modulatory oligonucleotides, IMO-2125 (IMO), significantly enhanced immune responses to hepatitis B virus surface antigen (HBsAg) and ovalbumin (OVA). LNPs enhanced total B-cell responses to both antigens tested, to levels comparable to known vaccine adjuvants including aluminum based adjuvant, IMO alone and a TLR4 agonist, 3-O-deactytaled monophosphoryl lipid A (MPL). Investigation of the quality of B-cell responses demonstrated that the combination of LNP with IMO agonist elicited a stronger Th1-type response (based on the IgG2a:IgG1 ratio) than levels achieved with IMO alone. Furthermore, the LNP adjuvant significantly enhanced antigen specific cell-mediated immune responses. In ELISPOT assays, depletion of specific subsets of T cells revealed that the LNPs elicited potent antigen-specific CD4⁺ and CD8⁺T cell responses. Intracellular FACS analyses revealed that LNP and LNP + IMO formulated antigens led to higher frequency of antigen-specific IFNγ⁺TNFα⁺IL-2⁺, multi-functional CD8⁺T cell responses, than unadjuvanted vaccine or vaccine with IMO only. Overall, our results demonstrate that lipid nanoparticles can serve as future sub-unit vaccine adjuvants to boost both B-cell and T-cell responses in vivo, and that addition of IMO can be used to manipulate the quality of immune responses.     

Using an effective TB vaccination regimen to identify immune responses associated with protection in the murine model

A vaccine against tuberculosis (TB), a disease resulting from infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus Calmette–Guérin (BCG) is the only available vaccine against TB but its efficacy varies throughout the world. Subunit vaccine candidates, based on recombinant viral vectors expressing mycobacterial antigens, are one of the strategies being developed to boost BCG-primed host immune responses and efficacy. A promising vaccination regimen composed of intradermal (i.d.) BCG prime, followed by intranasally (i.n.) administered chimpanzee adenoviral vector (ChAdOx1) and i.n. or i.d. modified vaccinia Ankara virus (MVA), both expressing Ag85A, has been previously reported to significantly improve BCG efficacy in mice. Effector and memory immune responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated to identify immune correlates of protection in mice. This protective regime induced strong Ag85A-specific cytokine responses in CD4⁺ and CD8⁺ T cells, both in the systemic and pulmonary compartments. Lung parenchymal CXCR3⁺ KLRG1^- Ag85A-specific memory CD4⁺ T cells were significantly increased in B-C-M compared to BCG immunised mice at 4, 8 and 20 weeks post vaccination, but the number of these cells decreased at the latter time point. This cell population was associated with the protective efficacy of this regime and may have an important protective role against M.tb infection.