Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.     

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP + M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8⁺, intracellular IFNγ⁺ cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.     

Induction of HIV-1-specific cellular and humoral immune responses following immunization with HIV-DNA adjuvanted with activated apoptotic lymphocytes

Delivery of DNA encoding foreign antigens into mammalian cells can induce adaptive immune responses. There are currently many DNA-based vaccines in clinical trials against infectious diseases and cancer but there is a lack of adjuvants for improvement of responses to DNA-based vaccines. Here, we show augmented systemic and mucosa-associated B cell responses after immunization with a cocktail of seven different plasmids (3 env, 2 gag, 1 rev, 1 RT) combined with mitogen activated apoptotic syngeneic lymphocytes in mice. In addition we show that apoptotic cells can function as adjuvant for induction of cellular immune responses in a magnitude comparable to the cytokine adjuvant GM-CSF in mice. These data suggest that activated apoptotic lymphocytes can act independent as adjuvants to improve antigen-specific DNA vaccines.     

Immunization and challenge experiments with a new modified live bovine herpesvirus type 1 marker vaccine prototype adjuvanted with a co-polymer

Western European control programs against bovine herpesvirus type 1 (BoHV-1) infections utilize attenuated BoHV-1 marker vaccines with a deletion of the glycoprotein E (gE) encoding gene. However, a recent study demonstrated the potential risk of virulence recovery of gE-deleted BoHV-1 marker vaccine strains due to recombination (Muylkens et al. [15]). Based on an infectious bacterial artificial chromosome clone, a gE- and thymidine kinase (TK)-gene-deleted BoHV-1 mutant (BoHV-1ΔgEΔTK) was constructed. The recombinant virus was subsequently tested as a novel modified live marker vaccine candidate in an immunization-challenge trial using BoHV-1 seronegative calves. Additionally, a non-virucidal co-polymer was tested together with the recombinant virus acting as a vaccine-adjuvant. Animals were vaccinated twice through intramuscular injection and challenged intranasally 3 weeks later with a virulent BoHV-1 field strain. Duration and titres of challenge virus shedding were significantly reduced in all vaccinees. Importantly, reduction of challenge virus shedding and serological antibody levels in response to vaccination with vaccine preparations containing the co-polymer-adjuvant were markedly improved when compared to vaccine formulations without an adjuvant. Taken together, our study describes a novel double deletion mutant as a safe and efficacious BoHV-1-prototype marker vaccine strain with enhanced protective capacity especially when administered together with a co-polymer adjuvant.     

Broadened immunity and protective responses with emulsion-adjuvanted H5 COBRA-VLP vaccines

A number of challenges for developing a protective pre-pandemic influenza A vaccine exists including predicting the target influenza strain and designing the vaccine for an immunologically naïve population. Manufacturing and supply of the vaccine would also require implementing ways to increase coverage for the largest number of people through dose-sparing methods, while not compromising the potency of the vaccine. Previously, our group described a novel hemagglutinin (HA) for H5N1 influenza derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This report describes a strategy combining a COBRA-based HA vaccine with an oil-in-water emulsion, resulting in a dose-sparing, immunologically broadened, and protective response against multiple H5N1 isolates. Here, we show that an emulsion-based adjuvant enhances the magnitude and breadth of antibody responses with both a wild-type H5HA (H5N1 WT) and the H5N1 COBRA HA VLP vaccines. The H5N1 COBRA HA VLP, combined with an emulsion adjuvant, elicited HAI specific antibodies against a larger panel of H5N1 viruses that resulted in protection against challenge as efficiently as the homologous, matched vaccine.     

Inflammatory parameters associated with systemic reactogenicity following vaccination with adjuvanted hepatitis B vaccines in humans

Background

Adjuvants like AS01_B increase the immunogenicity of vaccines and generally cause increased transient reactogenicity compared with Alum. A phase II randomized trial was conducted to characterize the response to AS01_B and Alum adjuvanted vaccines. A post-hoc analysis was performed to examine the associations between reactogenicity and innate immune parameters.

Local and systemic inoculation of DNA or protein gB1s-based vaccines induce a protective immunity against rabbit ocular HSV-1 infection

A secreted form of gB1 (gB1s), previously shown to protect rabbits against HSV-1 ocular infection when inoculated systemically, was delivered to rabbit periocular area to evaluate its vaccine efficacy upon local administration. The efficacy of local or systemic inoculation of a gB1s-DNA-based vaccine in the rabbit model of ocular HSV-1 infection was assessed in parallel flow. Rabbits received four inoculations of the different immunogens, then immune responses and clinical symptoms were evaluated. Both the local protein and the systemic DNA administration elicited a neutralizing antibody response, reduced ocular symptoms with respect to controls (P<0.01), and completely prevented the death of rabbits from encephalitis. Conversely, local DNA vaccination did not induce any detectable antibody response, and could only partially protect rabbits from the development of encephalitis and severe ocular infection.     

Protection against bovine respiratory syncytial virus in calves vaccinated with adjuvanted modified live vaccine administered in the face of maternal antibody

Bovine respiratory syncytial virus (BRSV) is major viral contributor to bovine respiratory disease (BRD). BRD is a major cause of morbidity and mortality in all classes of cattle but particularly young beef and dairy calves. Passive antibodies not only help protect the calf against infection, but may interfere with the immune responses following vaccination. The purpose of this study was to evaluate the efficacy of an adjuvanted modified live virus (MLV) vaccine in the presence of well-defined maternal passive immunity. Calves were vaccinated at approximately 1 month of age and challenged ~90 days later when BRSV systemic antibodies were ≤1:4. Body temperature was lower at 6 and 7 days post challenge and other clinical signs were also lower in the vaccinates. Nasal viral shed was 3–4 times lower in the vaccinated animals as measured by virus isolation and polymerase chain reaction (PCR) and peaked 5 days post challenge compared to the controls (who peaked at days 6 and 7). On day 8 following challenge, animals were necropsied, and lung lobes were scored and tested for virus by PCR and indirect fluorescent assay (IFA). There was a 25-fold reduction in PCR virus detection in vaccinates and two of the vaccinated calves’ lungs were PCR negative. Only 29.4% of vaccinated calves were BRSV positive on IFA testing at necropsy, while 87.5% of control calves were BRSV positive. Vaccinated calves developed a mucosal BRSV IgA response with over 50% of the vaccinated calves having IgA prior to challenge and all vaccinated calves were positive following challenge. Additionally, vaccination stimulated the production of Interferon gamma (IFN-γ) in mononuclear cells to prime the immune system. This study established that an adjuvanted MLV vaccine could provide protection against BRSV as measured by clinical, virological, and pathological parameters while also activating both mucosal and systemic immunity.     

Safety and immunogenicity of HCV E1E2 vaccine adjuvanted with MF59 administered to healthy adults

Background

Hepatitis C virus (HCV) causes chronic liver disease that often leads to cirrhosis and hepatocellular carcinoma. In animal studies, chimpanzees were protected against chronic infection following experimental challenge with either homologous or heterologous HCV genotype 1a strains which predominate in the USA and Canada. We describe the first in humans clinical trial of this prophylactic HCV vaccine.

Methods

HCV E1E2 adjuvanted with MF59C.1 (an oil-in-water emulsion) was given at 3 different dosages on day 0 and weeks 4, 24 and 48 in a phase 1, placebo-controlled, dose escalation trial to healthy HCV-negative adults.

Results

There was no significant difference in the proportion of subjects reporting adverse events across the groups. Following vaccination subjects developed antibodies detectable by ELISA, CD81 neutralization and VSV/HCV pseudotype neutralization. There were no significant differences between vaccine groups in the number of responders and geometric mean titers for each of the three assays. All subjects developed lymphocyte proliferation responses to E1E2 and an inverse response to increasing amounts of antigen was noted.

Conclusions

The vaccine was safe and generally well-tolerated at each of the 3 dosage levels and induced antibody and lymphoproliferative responses. A larger study to further evaluate safety and immunogenicity is warranted.     

Subunit vaccines with a saponin-based adjuvant boost humoral and cellular immunity to MERS coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks have constituted a public health issue with drastic mortality higher than 34%, necessitating the development of an effective vaccine. During MERS-CoV infection, the trimeric spike protein on the viral envelope is primarily responsible for attachment to host cellular receptor, dipeptidyl peptidase 4 (DPP4). With the goal of generating a protein-based prophylactic, we designed a subunit vaccine comprising the recombinant S1 protein with a trimerization motif (S1-Fd) and examined its immunogenicity and protective immune responses in combination with various adjuvants. We found that sera from immunized wild-type and human DPP4 transgenic mice contained S1-specific antibodies that can neutralize MERS-CoV infection in susceptible cells. Vaccination with S1-Fd protein in combination with a saponin-based QS-21 adjuvant provided long-term humoral as well as cellular immunity in mice. Our findings highlight the significance of the trimeric S1 protein in the development of MERS-CoV vaccines and offer a suitable adjuvant, QS-21, to induce robust and prolonged memory T cell response.     

Long-term evaluation of mucosal and systemic immunity and protection conferred by different polio booster vaccines

Oral polio vaccine (OPV) and Inactivated Polio Vaccine (IPV) have distinct advantages and limitations. IPV does not provide mucosal immunity and introduction of IPV to mitigate consequences of circulating vaccine-derived polio virus from OPV has very limited effect on transmission and OPV campaigns are essential for interrupting wild polio virus transmission, even in developed countries with a high coverage of IPV and protected sewer systems. The problem is magnified in many countries with limited resources. Requirement of refrigeration for storage and transportation for both IPV and OPV is also a major challenge in developing countries. Therefore, we present here long-term studies on comparison of a plant-based booster vaccine, which is free of virus and cold chain with IPV boosters and provide data on mucosal and systemic immunity and protection conferred by neutralizing antibodies.Mice were primed subcutaneously with IPV and boosted orally with lyophilized plant cells containing 1 μg or 25 μg polio viral protein 1 (VP1), once a month for three months or a single booster one year after the first prime. Our results show that VP1-IgG1 titers in single or double dose IPV dropped to background levels after one year of immunization. This decrease correlated with >50% reduction in seropositivity in double dose and <10% seropositivity in single dose IPV against serotype 1. Single dose IPV offered no or minimal protection against serotype 1 and 2 but conferred protection against serotype 3. VP1-IgA titers were negligible in IPV single or double dose vaccinated mice. VP1 antigen with two plant-derived adjuvants induced significantly high level and long lasting VP1-IgG1, IgA and neutralizing antibody titers (average 4.3–6.8 log2 titers). Plant boosters with VP1 and plant derived adjuvants maintained the same level titers from 29 to 400 days and conferred the same level of protection against all three serotypes throughout the duration of this study. Even during period, when no plant booster was given (∼260 days), VP1-IgG1 titers were maintained at high levels. Lyophilized plant cells expressing VP1 can be stored without losing efficacy, eliminating cold chain. Virus-free, cold-chain free vaccine is ready for further clinical development.     

A nasally administered trivalent inactivated influenza vaccine is well tolerated, stimulates both mucosal and systemic immunity, and potentially protects against influenza illness

A randomized placebo-controlled double-blind trial of a nasally administered inactivated trivalent influenza vaccine formulated with partially purified meningococcal outer membrane proteins (OMP-TIV) was conducted in 1349 healthy adults aged 18-64 years. Subjects received either vaccine containing 15 μg of haemagglutinin (HA) of each of three influenza strains for the 2003-2004 season on days 0 and 14, or 30 μg on day 0 and saline placebo on day 14, or placebo on days 0 and 14. Vaccination was well tolerated, with similar reactogenicity as placebo. Compared to placebo, statistically significant increases in mean serum haemagglutinin inhibition reciprocal titers and salivary secretory IgA to all 3 antigens were seen on day 28 for both vaccine dose groups. The incidence of culture-positive influenza and fever >37.8 °C and cough and one or more of sore throat, runny nose or nasal congestion, muscle or joint ache, headache, fatigue, or chills or culture positive influenza and at least two of these symptoms was low (16/1349; 1.2%). In the intent-to-immunize population too few febrile culture-confirmed illness events (n = 4) occurred to perform analysis. Fever occurred infrequently, even in the presence of positive cultures and disabling multi-symptom disease. In participants receiving all doses of either vaccine regimen the incidence of culture-confirmed influenza with respiratory symptoms and with or without fever was 0.77% (7/904) vs. 2.03% (9/443) in placebo recipients (p = 0.045, Fisher's exact test; relative risk reduction 62%), despite circulation of a drift variant A/H3N2 that was poorly matched to vaccine. An OMP-TIV vaccine was well tolerated and reduced risk of symptomatic culture confirmed influenza. Vaccine efficacy will need to be validated in a season with a higher attack rate.     

Post-fusion influenza vaccine adjuvanted with SA-2 confers heterologous protection via Th1-polarized,non-neutralizing antibody responses

Development of a universal influenza vaccine that can provide robust and long-lasting protection against heterologous infections is a global public health priority. A variety of vaccine antigens are designed to increase the antigenicity of conserved epitopes to elicit cross-protective antibodies that often lack virus-neutralizing activity. Given the contribution of antibody effector functions to cross-protection, adjuvants need to be added to modulate antibody effector functions as well as to enhance antibody quantity. We previously showed that post-fusion influenza vaccine antigens elicit non-neutralizing but cross-protective antibodies against conserved epitopes. Here, using a murine model, we comparably assessed the adjuvanticity of the newly developed SA-2 adjuvant containing a synthetic TLR7 agonist DSP-0546 and squalene-based MF59 analog as representative Th1- or Th2-type adjuvants, respectively. Both types of adjuvants in the post-fusion vaccine comparably enhanced cross-reactive IgG titers against heterologous strains. However, only SA-2 skewed the IgG subclass into the IgG2c subclass in association to its Th1-polarizing nature. SA-2-enhanced IgG2c responses exhibited antibody-dependent cellular cytotoxicity against heterologous virus strains, without cross-neutralizing activity. Eventually, the SA-2-adjuvanted vaccination provided protection against lethal infection by heterologous H3N2 and H1N1 viruses. Together, we conclude that the combination with a SA-2 is advantageous for enhancing the cross-protective capability of post-fusion HA vaccines that elicit non-neutralizing IgG antibodies.     

Cross-clade protective immunity of H5N1 influenza vaccines in a mouse model

H5N1 highly pathogenic avian influenza viruses evolved into several clades, leading to appreciably distinct antigenicities of their hemagglutinins. As such, candidate H5N1 pre-pandemic vaccines for human use should be sought. Here, to evaluate fundamental immunogenic variations between H5N1 vaccines, we prepared four inactivated H5N1 test vaccines from different phylogenetic clades (clade 1, 2.1, 2.2, and 2.3.4) in accordance with the WHO recommendation, and tested their cross-clade immunity in a mouse model by vaccination followed by challenge with heterologous virulent viruses. All H5N1 vaccines tested provided full or partial cross-clade protective immunity, except one clade 2.2-based vaccine, which did not protect mice from clade 2.3.4 virus challenge. Among the test vaccines, a clade 2.1-based vaccine possessed the broadest-spectrum cross-immunity. These results suggest that currently stockpiled pre-pandemic vaccines, especially clade 2.1-based vaccines, will likely be useful as backup vaccines in a pandemic situation, even one involving antigenic-drifted viruses.     

Superior immunogenicity of HCV envelope glycoproteins when adjuvanted with cyclic-di-AMP,a STING activator or archaeosomes

Three decades after the discovery, hepatitis C virus (HCV) is still the leading cause of liver transplantation and poses a major threat to global health. In spite of recent advances in the development of direct acting antivirals, there is still a need for a prophylactic vaccine to limit the virus spread and protect at-risk populations, especially in developing countries, where the cost of the new treatments may severely limit access. The use of recombinant HCV glycoproteins E1E2 (rE1E2) in combination with the MF59, an oil-in-water emulsion-based adjuvant, has previously been shown to reduce the rate of chronicity in chimpanzees and to induce production of cross-neutralizing antibodies and cellular immune responses in human volunteers. To further improve neutralizing antibody responses in recipients along with robust T cell responses, we have explored the immunogenicity of different adjuvants when formulated with the HCV rE1E2 vaccine in mice.Our data show that cyclic di-adenosine monophosphate (c-di-AMP) and archaeosomes elicit strong neutralizing antibodies similar to those elicited using aluminum hydroxide/monophosphoryl lipid A (Alum/monophos. /MPLA) and MF59. However, both c-di-AMP and archaeosomes induced a more robust cellular immune response, which was confirmed by the detection of vaccine-specific poly-functional CD4⁺ T cells. We conclude that these adjuvants may substantially boost the immunogenicity of our E1E2 vaccine. In addition, our data also indicates that use of a partial or exclusive intranasal immunization regimen may also be feasible using c-di-AMP as adjuvant.     

Vaccine adjuvant activity of 3M-052: an imidazoquinoline designed for local activity without systemic cytokine induction

The human Toll-like receptors (TLRs) are a family of receptors, which sense the presence of various structural elements of pathogens and damaged or effete components in the host. As they do so, they activate two critical arms of host defense, the rapid innate immune response and an adaptive immune response. The innate immune response is typified by the generation of Th1 cytokines, chemokines and type 1 interferons. As such, agonists for the TLRs have potential as antiviral and anticancer therapeutics. They are also well suited to function as vaccine adjuvants. 3M imidazoquinoline (IRM) molecules were the first synthetic small molecules identified as TLR agonists and can affect their biological activities through TLR7, TLR8, or both. The breadth of therapeutic opportunities for this family of molecules can require formulations tailored to the specific application. One consideration is specific formulations to avoid a systemic distribution of these TLR agonists and resulting cytokine storm-like effects on the host. 3M-052 is an IRM bearing a C18 lipid moiety and designed for slow dissemination from the site of application. In the present study 3M-052 has been evaluated for its in vitro TLR activity and for its efficacy as a vaccine adjuvant using a recombinant hemagglutinin from H1N1 A/Puerto Rico/8/34. Given subcutaneously, 3M-052 drives a strong Th1 response to hemagglutinin and serum neutralization of viable H1N1 A/Puerto Rico/8/34 virus in the absence of circulating TNFα or the induction of Th1 cytokines.     

Enhanced humoral and Type 1 cellular immune responses with Fluzone adjuvanted with a synthetic TLR4 agonist formulated in an emulsion

Impairments in anti-influenza T helper 1 (Th1) responses are associated with greater risk of influenza-related mortality in the elderly. Addition of adjuvants to existing influenza vaccines could improve immune responses in the elderly. In this study, the activity of three adjuvants, an oil-in-water emulsion and a synthetic lipid A adjuvant formulated with or without the emulsion, is compared. Our results show that Fluzone combined with lipid A plus an emulsion effectively leads to greater vaccine-specific IgG2a and IgG titers, enhances hemagglutination-inhibition titers and induces Type 1 cytokine responses (IFN-γ and IL-2) to each of the Fluzone components.     

The induction of systemic and mucosal immunity to protein vaccines delivered through skin sites exposed to UVB

It has been reported that common mucosal immunity can be efficiently induced in mice following immunization through the skin with vaccine formulations containing either the active form of vitamin D, or chemical agents capable of locally enhancing cyclic adenosine monophosphate levels. Herein, we report that exposure of skin to ultraviolet radiation B (UVB) can be employed as a means to alter systemic humoral immune responses and to promote the induction of mucosal immunity to protein antigens delivered into UVB-exposed skin sites. Our data indicates that the skin, as a vaccination site, can be manipulated to allow efficient induction of common mucosal and systemic immune responses.     

Enhanced humoral and cell-mediated immune responses after immunization with trivalent influenza vaccine adjuvanted with cationic liposomes

The recent pandemic caused by new influenza A (H1N1) has emphasized the need for improved influenza vaccines with enhanced immune responses that ideally include longlived humoral and CMI responses and mediate a broad protection. This study demonstrates that administration of trivalent influenza vaccine (TIV) with the cationic liposome adjuvant system CAF01 enhances the humoral immune response as measured by hemagglutinin inhibition titers and influenza-specific serum antibody titers, and promote a strong Th1 response with augmented levels of IL-1β, IL-2, IL-12, IFN-γ and TNF-α. Furthermore, high levels of IL-17 are detected in agreement with CAF01's ability to promote TH17 responses. Importantly, the Th1/Th17 cytokine profile is still maintained 20 weeks after the last vaccination. The CAF01 adjuvanted influenza vaccine reduces weight loss and temperature decrease and results in complete survival of mice challenged with the drifted H1N1 influenza strain A/PR/8/34. Overall, the results suggest that CAF01 is a potent adjuvant system for future, improved influenza vaccines.