Recombinant outer membrane vesicles to augment antigen-specific live vaccine responses

Release of outer membrane vesicles is a common feature of Gram negative bacteria. There is growing interest in the use of these vesicles in the development of affordable vaccines. However, to exploit their full potential a convenient system to generate recombinant vesicles would be highly desirable. Here, we report the design of a versatile system for preparation of recombinant outer membrane vesicles based on an engineered autotransporter. Two model vaccine antigens of Leishmania were expressed as fusion proteins with the transporter domain of AIDA, the Escherichia coli adhesin involved in diffuse adhesion. Single subcutaneous injections of recombinant vesicles boosted vaccine antigen-specific antibody responses in mice primed with live recombinant salmonella vaccines by 6–40-fold. The results further show an expansion of only the vaccine antigen-specific antibody response indicating a great potential of this approach for prime boost vaccination strategies.     

UTI patients have pre-existing antigen-specific antibody titers against UTI vaccine antigens

Urinary tract infection (UTI) is most frequently caused by uropathogenic Escherichia coli (UPEC). Our laboratory has been developing an experimental vaccine targeting four UPEC outer membrane receptors involved in iron acquisition – IreA, FyuA, IutA, and Hma – to elicit protection against UTI. These vaccine targets are all expressed in humans during UTI. In the murine model, high titers of antigen-specific serum IgG or bladder IgA correlate with protection against transurethral challenge with UPEC. Our aim was to measure levels of pre-existing serum antibodies to UTI vaccine antigens in our target population. To accomplish this, we obtained sera from 64 consenting female patients attending a clinic for symptoms of cystitis. As a control, we also collected sera from 20 healthy adult male donors with no history of UTI. Total IgG and antigen-specific IgG titers were measured by ELISA. Of the 64 female patients, 29 had significant bacteriuria (>10⁴ cfu/ml urine) and uropathogenic E. coli (UPEC). Thirty-five patients had non-significant bacteriuria (<10⁴ cfu/ml). Antigen-specific IgG titers did not correlate with the presence or absence of the gene encoding the antigen in the infecting strain (when present), but rather titers were proportional to prevalence of genes encoding antigens among representative collections of UPEC isolates. Surprisingly, we obtained similar results when sera from healthy male patients without history of UTI were tested. Thus, unvaccinated adults have non-protective levels of pre-existing antibodies to UTI vaccine antigens, establishing an important baseline for our target population. This suggests that a UTI vaccine would need to boost pre-existing humoral responses beyond these background levels to protect from infection.     

Measuring antigen-specific bactericidal responses to a multicomponent vaccine against serogroup B meningococcus

Serum bactericidal activity using human complement is the basis for established correlates of protection against invasive meningococcal disease. During the development of multicomponent protein-based vaccines against meningococcus B, it is necessary to measure antigen-specific bactericidal responses. This is not straightforward because each strain may be killed by antibodies to multiple antigens. We characterized a large panel of strains and, using a competitive inhibition SBA, we identified four strains that are each specifically killed by bactericidal antibodies to one of the major vaccine components. These strains provide a straightforward approach to demonstrate protective responses to each component of the vaccine and demonstrate that each of the antigens in the vaccine is sufficient to provide a potentially protective level of bactericidal activity.     

Homologous and heterologous antibody responses to subunit influenza virus vaccine

In a group of 32 adult volunteers given subunit influenza virus vaccine containing 250 international units (i.u.) of A/Victoria/3/75, 250 i.u. of A/Scotland/840/74 and 300 i.u. of B/Hong Kong/8/73, there were substantial increases in the geometric mean homologous haemagglutination-inhibiting (HI) antibody titres. There was also substantial boosting of the antibodies to the earlier variants of the Hong Kong (H3N2) series and to a later variant of the Asian (H2N2) series. There was no boosting of antibodies to the A/FM/1/47 strain, a representative member of the H1N1 series, but two individuals showed substantial rises to A/PR/8/34 (HON1). There were increases in the HI titre of antibodies cross reactive with two recent isolations, A/Texas/1/77, and A/Victoria/35/77, but the majority of vaccinees failed to reach antibody titres likely to be protective against such strains.     

Interleukin-6 has differential influence on the ability of adjuvant formulations to potentiate antibody responses to a Plasmodium falciparum blood-stage vaccine

The efficacy of vaccine adjuvants can be influenced by the immunological environment of the host, depending on the mechanism(s) by which they exert their immunopotentiating activities. Interleukin-6 is a pleiotropic cytokine that has a broad range of biological activities on immune and non-immune cells. We investigated the role of IL-6 on the ability of nine adjuvant formulations to induce antibody responses to the Plasmodium falciparum MSP1-19 malaria vaccine, using IL-6-/- (KO) mice. Results showed that some adjuvants, i.e. MPL-SE, CFA/IFA, ISA720/QS21/MPL, depended on IL-6 for their efficacy, while others exhibited increased potency in its absence. The efficacy of adjuvants in the IL-6 KO environment cannot be solely attributed to their ability to stimulate antigen-specific cellular responses, suggesting that other biological activities of IL-6 are also important. The results further suggest that two adjuvants utilized dissimilar pathways to potentiate the same type of immune response.     

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.     

A new adjuvanted nanoparticle-based H1N1 influenza vaccine induced antigen-specific local mucosal and systemic immune responses after administration into the lung

Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed.     

Homologous and heterologous antibody responses to subunit influenza virus vaccine.

In a group of 32 adult volunteers given subunit influenza virus vaccine containing 250 international units (i.u.) of A/Victoria/3/75, 250 i.u. of A/Scotland/840/74 and 300 i.u. of B/Hong Kong/8/73, there were substantial increases in the geometric mean homologous haemagglutination-inhibiting (HI) antibody titres. There was also substantial boosting of the antibodies to the earlier variants of the Hong Kong (H3N2) series and to a later variant of the Asian (H2N2) series. There was no boosting of antibodies to the A/FM/1/47 strain, a representative member of the H1N1 series, but two individuals showed substantial rises to A/PR/8/34 (HON1). There were increases in the HI titre of antibodies cross reactive with two recent isolations, A/Texas/1/77, and A/Victoria/35/77, but the majority of vaccinees failed to reach antibody titres likely to be protective against such strains.     

Mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-DNA vaccine in a gnotobiotic pig model

A live rotavirus prime/DNA boost vaccine regimen was evaluated in a gnotobiotic pig model for human rotavirus (HRV) diarrhea. Plasmid DNA expressing rotavirus inner capsid VP6 was administered to pigs intramuscularly (IM) twice after oral priming with attenuated (Att) Wa strain HRV (AttHRV/VP6DNA2x). Other groups included: (1) VP6 DNA IM 2x then AttHRV orally (VP6DNA2x/AttHRV); (2) VP6 DNA IM 3x (VP6DNA3x) and controls. Significant protection (70%) against virus shedding, but lower protection against diarrhea (30%) was achieved only in the AttHRV/VP6DNA2x group after challenge (virulent Wa HRV). The other vaccines (VP6DNA2x/AttHRV and VP6DNA3x) were less effective. Higher protection rates were associated with the highest IgA antibody responses induced by the AttHRV/VP6DNA2x regimen. Interestingly, the VP6 DNA vaccine, although not effective when administered alone, boosted neutralizing and VP4 antibody titers in pigs previously primed with AttHRV, possibly mediated by cross-reactive T helper cells.     

Immune markers and correlates of protection for vaccine induced immune responses

Vaccines have been a major innovation in the history of mankind and still have the potential to address the challenges posed by chronic intracellular infections including tuberculosis, HIV and malaria which are leading causes of high morbidity and mortality across the world. Markers of an appropriate humoral response currently remain the best validated correlates of protective immunity after vaccination. Despite advancements in the field of immunology over the past few decades currently there are, however, no sufficiently validated immune correlates of vaccine induced protection against chronic infections in neither human nor veterinary medicine. Technological and conceptual advancements within cell-mediated immunology have led to a number of new immunological read-outs with the potential to emerge as correlates of vaccine induced protection. For T(H)1 type responses, antigen-specific production of interferon-gamma (IFN-γ) has been promoted as a quantitative marker of protective cell-mediated immune responses over the past couple of decades. More recently, however, evidence from several infections has pointed towards the quality of the immune response, measured through increased levels of antigen-specific polyfunctional T cells capable of producing a triad of relevant cytokines, as a better correlate of sustained protective immunity against this type of infections. Also the possibilities to measure antigen-specific cytotoxic T cells (CTL) during infection or in response to vaccination, through recombinant major histocompatibility complex (MHC) class I tetramers loaded with relevant peptides, has opened a new vista to include CTL responses in the evaluation of protective immune responses. Here, we review different immune markers and new candidates for correlates of a protective vaccine induced immune response against chronic infections and how successful they have been in defining the protective immunity in human and veterinary medicine.     

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of Human Immunodeficiency Virus Type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial conducted in healthy adult volunteers of both genders. Robust cross-subtype HIV-1-specific T cell responses were detected in IFNgamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.     

Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers

An optimally effective AIDS vaccine would likely require the induction of both neutralizing antibody and cell-mediated immune responses, which has proven difficult to obtain in previous clinical trials. Here we report on the induction of human immunodeficiency virus type-1 (HIV-1)-specific immune responses in healthy adult volunteers that received the multi-gene, polyvalent, DNA prime-protein boost HIV-1 vaccine formulation, DP6-001, in a Phase I clinical trial. Robust cross-subtype HIV-1 specific T cell responses were detected in IFN-gamma ELISPOT assays. Furthermore, we detected high titer serum antibody responses that recognized a wide range of primary HIV-1 Env antigens and also neutralized pseudotyped viruses that express the primary Env antigens from multiple HIV-1 subtypes. These findings demonstrate that the DNA prime-protein boost approach is an effective immunization method to elicit both humoral and cell-mediated immune responses in humans, and that a polyvalent Env formulation could generate broad immune responses against HIV-1 viruses with diverse genetic backgrounds.     

The effect of mucoadhesive excipient on the nasal retention time of and the antibody responses induced by an intranasal influenza vaccine

IntroductionRecently, we reported that intranasal vaccination of humans with whole inactivated influenza vaccine in the absence of mucosal adjuvant induced neutralizing antibody responses in the serum and nasal mucus. The mucoadhesive excipient carboxy-vinyl polymer (CVP) increases the viscosity and therefore mucoadhesiveness of intranasal medicaments and is an authorized excipient in Japan. In the present study, we analyzed the effect of adding CVP on intranasal whole inactivated influenza vaccine antigen dynamics and antibody responses.MethodsMice and nonhuman primates (NHPs) were intranasally administered the [¹⁸F]-radiolabeled vaccine and subjected to positron emission tomography analysis for 6 h. Dendritic cells were stimulated in vitro with the vaccine mixed with or without a mucosal adjuvant (Ampligen) and/or CVP, after which the tumor necrosis factor (TNF)-α and interferon (IFN)-β levels in the supernatants were measured. Cynomolgus monkeys were immunized intranasally with the vaccine mixed with Ampligen and/or CVP and their vaccine-specific serum IgG and IgA titers were measured on days 0 and 33.ResultsThe vaccine was retained significantly longer in the nasal cavity of both mice and NHPs when it was delivered with CVP rather than PBS. Accumulation of the radiolabeled vaccine in the central nervous system was not detected in either model regardless of whether CVP was used. CVP only very weakly increased the TNF-α production of vaccine-stimulated dendritic cells. IFN-β production was not observed regardless of the presence or absence of CVP. CVP increased the vaccine-specific IgA antibody responses of the intranasally vaccinated cynomolgus macaques.ConclusionCVP increased intranasal retention of whole inactivated influenza vaccine, did not promote antigen redirection to the central nervous system, and improved mucosal antibody responses. The mechanism probably relates to its mucoadhesive properties rather than its ability to directly stimulate the immune system. Intranasal vaccines with CVP may be a promising candidate vaccine formulation for humans.     

Mucosal antibody response induced with a nasal virosome-based influenza vaccine

A vaccination against influenza that elicits both a systemic antibody and a mucosal IgA response would improve on the protective efficacy of currently available vaccines. Previous studies have shown the safety and efficacy of virosomes as delivery systems in vaccination. This study was a controlled, randomised, double-blind, single centre, phase II trial assessing an intranasal virosome vaccine, adjuvanted with heat-labile toxin (HLT) from enterotoxigenic Escherichia coli, versus an intranasal without HLT and comparing it open to an intramuscular vaccine in a total of 88 healthy adults. The development of a new technique enabled for the first time the detection of neutralising IgA antibodies in very dilute nasal wash samples. It was demonstrated that intranasally administered inactivated influenza vaccine, adjuvanted with HLT, not only elicits a spectrum of humoral and cell-mediated responses in healthy adults, critical for the protection and recovery from influenza virus infection, but is also highly effective in eliciting IgA neutralising antibodies at the mucosa. Intranasal virosome-formulated, HLT-adjuvanted, influenza vaccine was effective and well tolerated in this study. Its potential to offer a high level of mucosal protection, not provided by conventional parenteral vaccination, could play a significant role in preventing morbidity and mortality associated with influenza.     

Infants with low vaccine antibody responses have altered innate cytokine response

We recently identified a population of 10% of infants who respond with sub-protective antibody levels to most routine primary pediatric vaccinations due to altered innate and adaptive immune responses. We term these infants as low vaccine responders (LVRs). Here we report new data showing that TLR7/8 agonist – R848 stimulation of PBMCs of LVR infants elicit significantly lower IFN-α, IL-12p70 and IL-1β, while inducing higher levels of CCL5 (RANTES) compared to normal vaccine responder (NVR) infants.     

A multivalent polyomavirus vaccine elicits durable neutralizing antibody responses in macaques

In 2019, there were about 100,000 kidney transplants globally, with more than a quarter of them performed in the United States. Unfortunately, some engrafted organs are lost to polyomavirus-associated nephropathy (PyVAN) caused by BK and JC viruses (BKPyV and JCPyV). Both viruses cause brain disease and possibly bladder cancer in immunosuppressed individuals. Transplant patients are routinely monitored for BKPyV viremia, which is an accepted hallmark of nascent nephropathy. If viremia is detected, a reduction in immunosuppressive therapy is standard care, but the intervention comes with increased risk of immune rejection of the engrafted organ. Recent reports have suggested that transplant recipients with high levels of polyomavirus-neutralizing antibodies are protected against PyVAN. Virus-like particle (VLP) vaccines, similar to approved human papillomavirus vaccines, have an excellent safety record and are known to induce high levels of neutralizing antibodies and long-lasting protection from infection. In this study, we demonstrate that VLPs representing BKPyV genotypes I, II, and IV, as well as JCPyV genotype 2 produced in insect cells elicit robust antibody titers. In rhesus macaques, all monkeys developed neutralizing antibody titers above a previously proposed protective threshold of 10,000. A second inoculation, administered 19 weeks after priming, boosted titers to a plateau of $\ge$ 25,000 that was maintained for almost two years. No vaccine-related adverse events were observed in any macaques. A multivalent BK/JC VLP immunogen did not show inferiority compared to the single-genotype VLP immunogens. Considering these encouraging results, we believe a clinical trial administering the multivalent VLP vaccine in patients waiting to receive a kidney transplant is warranted to evaluate its ability to reduce or eliminate PyVAN.     

MAS-1, a novel water-in-oil adjuvant/delivery system,with reduced seasonal influenza vaccine hemagglutinin dose may enhance potency,durability and cross-reactivity of antibody responses in the elderly

BackgroundIncreased influenza vaccine efficacy is needed in the elderly at high-risk for morbidity and mortality due to influenza infection. Adjuvants may allow hemagglutinin (HA) dose-sparing with enhanced immunogenicity. MAS-1 is an investigational water-in-oil emulsion-based adjuvant/delivery system comprised of stable nanoglobular aqueous droplets.MethodsA phase 1, randomized, double-blind, safety and immunogenicity, adjuvant dose escalation trial was conducted in persons aged 65 years and older. MAS-1 adjuvant dose volumes at 0.3 mL or 0.5 mL containing 9 µg per HA derived from licensed seasonal trivalent influenza vaccine (IIV, Fluzone HD 60 µg per HA, Sanofi Pasteur) were compared to high dose (HD) IIV (Fluzone HD). Safety was measured by reactogenicity, adverse events, and safety laboratory measures. Immunogenicity was assessed by serum hemagglutination inhibition (HAI) antibody titers.ResultsForty-five subjects, aged 65–83 years, were randomly assigned to receive 9 µg per HA in 0.3 mL MAS-1 (15 subjects) or HD IIV (15 subjects) followed by groups randomly assigned to receive 9 µg per HA in 0.5 mL MAS-1 (10 subjects) or HD IIV (5 subjects). Injection site tenderness, induration, and pain, and headache, myalgia, malaise and fatigue were common, resolving before day 14 post-vaccination. Clinically significant late-onset injection site reactions occurred in four of ten subjects at the 0.5 mL adjuvant dose. Safety laboratory measures were within acceptable limits. MAS-1-adjuvanted IIV enhanced mean antibody titers, mean-fold increases in antibody titer, and seroconversion rates against vaccine strains for at least 168 days post-vaccination and enhanced cross-reactive antibodies against some non-study vaccine influenza viruses.ConclusionMAS-1 adjuvant provided HA dose-sparing without safety concerns at the 0.3 mL dose, but the 0.5 mL dose caused late injection site reactions. MAS-1-adjuvanted IIV induced higher HAI antibody responses with prolonged durability including against historical strains, thereby providing greater potential vaccine efficacy in the elderly throughout an influenza season.Clinical Trial Registry: ClinicalTrials.gov # NCT02500680.     

Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine

Injecting mice with killed cells of non-capsulated strain RM200 adsorbed on Al(OH)3 (pneumococcal whole-cell vaccine; WCV) reduces nasopharyngeal colonization by capsular serotype 6B and prevents fatal aspiration pneumonia by serotype 3 or serotype 5 strains. To further examine the potential for omni-strain immunity, we here examined a panel of clinical isolates and a library of capsule-switch variants in the TIGR4 background. IgG binding to these bacteria in sera of rabbits injected with WCV or Al(OH)3 alone was assayed by ELISA without and with adsorption with cell-wall polysaccharide, a species-common antigen. The examined strains were 23 primary isolates including at least 10 different MLS types and 13 serotypes; 15 of these strains were invasive isolates, subsequently mouse-passed. Additionally, to investigate the effect of capsulation, TIGR4 strain constructs with the capsulation genes of 20 different serotypes were evaluated. In ELISA all strains showed a large difference in IgG binding due to the immunization, of which most of the antibody typically was not CWPS-adsorbed and presumably directed to exposed protein antigens. Increased binding of IgG in the WCV-immunized serum to the 20 isogenic capsule-switch strains was shown also by flow cytometry. Further, all these 20 strains elicited IL-17A in T cells of WCV-vaccinated mice, a cytokine known to accelerate pneumococcal clearance. Thus WCV induced both humoral and T(H)17 cell-mediated immunity against all tested strains.     

Aerosol and subcutaneous measles vaccine: measles antibody responses 6 years after re-vaccination

There are no large-scale data on the long-term persistence of measles antibody after vaccination by the aerosol route. We therefore followed-up South African schoolchildren 6 years after their re-vaccination with Edmonston-Zagreb (EZ) and Schwarz (SW) measles vaccine given by aerosol and subcutaneous routes. Measles antibody levels and the proportion of children who were seropositive at year 6 remained significantly higher in the Edmonston-Zagreb aerosol group compared to the groups that received Schwarz or Edmonston-Zagreb vaccine subcutaneously. Measles re-vaccination by aerosol evokes a stronger and much longer lasting antibody response than injected vaccine and should thus provide more durable protection against measles.