Theresa Fitzgerald

Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults

BACKGROUND The need for worldwide seasonal and pandemic vaccine production has increased interest in the development of innovative technologies for influenza vaccine production. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in E. coli. METHODS 128 healthy adult subjects 18-49 years old were enrolled in a clinical trial conducted in three stages at a single center. Stage 1 was an open-label, dose escalation study in which the VAX125 vaccine was administered intramuscularly (im) at doses of 0.1 μg, 0.3 μg, 1 μg, 2 μg, 3 μg, 5 μg and 8 μg to groups of 8 subjects each. Stage 2 was a double-blind, placebo-controlled study in which subjects were randomized to receive 1.0 μg and 2.0 μg VAX125 vaccine doses or placebo, with 16 subjects per group. Finally, an additional 24 subjects received a 0.5 μg dose of VAX125 in stage 3, which was a non-randomized, open label study. In all parts subjects were followed for adverse events and sera was tested by hemagglutination-inhibition (HAI) and microneutralization (MN) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP), cytokine levels, and anti-flagellin antibody were also assessed. RESULTS Vaccine was generally well tolerated and there were no serious adverse events. Pain at the injection site was the most common local adverse event, and was mild or moderate in intensity. Systemic symptoms after vaccination include fatigue and headache, and two subjects, who received either 3 or 8 μg, had moderately severe systemic symptoms accompanied by substantial increases in serum CRP. Serum antibody responses against SI were seen by HAI and MN in most study subjects, with the geometric mean titer of post vaccination antibody increasing in a dose-dependent fashion. Overall, four-fold or greater serum HAI responses were seen in 61 of 96 (64%) subjects who received doses of 0.5 μg or greater, including in 46 of 72 subjects who received doses from 0.5 μg to 2 μg. CONCLUSIONS The globular head of the influenza HA expressed in a prokaryotic system was able to induce a functional antibody response against native virions. Vigorous responses were seen at relatively low doses of HA antigen suggesting that the addition of flagellin provided a substantial adjuvanting effect. The high levels of immune response at low doses of antigen and the relative ease of production associated with E. coli expression suggests that this approach may represent an effective strategy for enhancing the global influenza vaccine supply.

Induction of a potent immune response in the elderly using the TLR-5 agonist, flagellin, with a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125, STF2.HA1 SI).

BACKGROUND Influenza vaccines perform poorly in the elderly with reduced serological response and vaccine efficacy. We evaluated a novel influenza vaccine consisting of the globular head of the HA1 domain of the A/Solomon Islands/3/2006 (H1N1) influenza virus (VAX125) genetically fused to the TLR5 ligand, flagellin, and produced in Escherichia coli. METHODS 120 subjects ≥ 65 years old were enrolled at three clinical centers. VAX125 vaccine was administered at doses of 0.5, 1, 2, 3, 5 or 8 μg delivered i.m. as a single dose vaccination on Day 0 using a dose-escalation with 20 subjects in each dose level. Subjects were followed for adverse events and sera were tested by hemagglutination-inhibition (HAI) against egg-grown virus on days 0, 7, 14, and 28. Serum C-reactive protein (CRP) and anti-flagellin antibody were also assessed. RESULTS The mean age was 71 years. The vaccine was well tolerated at all dose levels, with no more than mild to moderate local or systemic symptoms. The geometric mean titers (GMT) increased in all dose groups. In the 5 μg group the day 14 post-vaccination HAI titer was 1:226 showing a 12-fold increase over baseline. The 8 μg group showed a similar post-vaccination GMT increase (∼ 8-fold). In the combined 5 and 8 μg groups, the seroconversion rate was 75% and the seroprotection rate was 98%. CONCLUSIONS A 5 μg dose of VAX125 was safe and able to induce a greater than 10-fold increase HAI antibody levels and nearly complete seroprotection in subjects over 65 years old. The use of flagellin to adjuvant influenza vaccines via the TLR5 innate immune pathway appears to be a useful approach to overcome poor immune responses in the elderly. VAX125 is a promising new candidate for prevention of influenza A disease in both young adults and the elderly.

Highly active antiretroviral therapy results in a decrease in CD8+ T cell activation and preferential reconstitution of the peripheral CD4+ T cell population with memory rather than naive cells

OBJECTIVE Highly active antiretroviral therapy (HAART) can produce marked increases in peripheral blood CD4+ T cells and decreases in HIV plasma RNA copy numbers. However, it is not clear whether these absolute changes will be accompanied by a recovery in the known naive CD4+ T cell depletion or a decrease in the marked CD8+ T cell activation. DESIGN Twenty-nine patients were enrolled in studies of either nucleoside therapy alone or nucleoside therapy combined with a protease inhibitor (zidovudine + lamivudine + indinavir). One hundred and ninety-one examinations were carried out at three baseline time points and during 40 weeks of follow-up to evaluate the effect of HAART on CD4+ memory/naive phenotype and CD8+ T cell activation. METHODS CD4+ and CD8+ T cell number, CD62L/CD45RA expression on CD4+ T cells and CD38 expression on CD8+ T cells were measured by three-color flow cytometry. RESULTS Most protease inhibitor treated patients had a significant rise in CD4+ numbers. The marked rise in the CD4+ T cells seen in individuals in this study was not accompanied over a 40-week period by a change in the abnormally low CD4+ naive compartment, and thus was almost completely of memory phenotype. The CD38 expression on CD8+ cells fell during treatment, and decreased to a greater degree than the comparable rise in CD4+ T cell counts. This decrease continued in many patients after the CD4+ T cell rise or viral load decline had plateaued. CONCLUSION HAART results in changes in activation to a greater extent than absolute changes in CD4+ T cell numbers, but is not accompanied by an increase in naive CD4+ T cells. Measurements of CD4+ T cell numbers alone may not allow appropriate interpretation of immune activation or immune competence in patients receiving those drugs.

B Cell Response and Hemagglutinin Stalk-Reactive Antibody Production in Different Age Cohorts following 2009 H1N1 Influenza Virus Vaccination

ABSTRACT The 2009 pandemic H1N1 (pH1N1) influenza virus carried a swine-origin hemagglutinin (HA) that was closely related to the HAs of pre-1947 H1N1 viruses but highly divergent from the HAs of recently circulating H1N1 strains. Consequently, prior exposure to pH1N1-like viruses was mostly limited to individuals over the age of about 60 years. We related age and associated differences in immune history to the B cell response to an inactivated monovalent pH1N1 vaccine given intramuscularly to subjects in three age cohorts: 18 to 32 years, 60 to 69 years, and ≥70 years. The day 0 pH1N1-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers were generally higher in the older cohorts, consistent with greater prevaccination exposure to pH1N1-like viruses. Most subjects in each cohort responded well to vaccination, with early formation of circulating virus-specific antibody (Ab)-secreting cells and ≥4-fold increases in HAI and MN titers. However, the response was strongest in the 18- to 32-year cohort. Circulating levels of HA stalk-reactive Abs were increased after vaccination, especially in the 18- to 32-year cohort, raising the possibility of elevated levels of cross-reactive neutralizing Abs. In the young cohort, an increase in MN activity against the seasonal influenza virus A/Brisbane/59/07 after vaccination was generally associated with an increase in the anti-Brisbane/59/07 HAI titer, suggesting an effect mediated primarily by HA head-reactive rather than stalk-reactive Abs. Our findings support recent proposals that immunization with a relatively novel HA favors the induction of Abs against conserved epitopes. They also emphasize the need to clarify how the level of circulating stalk-reactive Abs relates to resistance to influenza.

CD4+ T-cell expansion predicts neutralizing antibody responses to monovalent, inactivated 2009 pandemic influenza A(H1N1) virus subtype H1N1 vaccine.

Abstract Background. The ability of influenza vaccines to elicit CD4+ T cells and the relationship between induction of CD4+ T cells and vaccine-induced neutralizing antibody responses has been controversial. The emergence of swine-origin 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) provided a unique opportunity to examine responses to an influenza vaccine composed of both novel and previously encountered antigens and to probe the relationship between B-cell and T-cell responses to vaccination. Methods. We tracked CD4+ T-cell and antibody responses of human subjects vaccinated with monovalent subunit A(H1N1)pdm09 vaccine. The specificity and magnitude of the CD4+ T-cell response was evaluated using cytokine enzyme-linked immunosorbent spot assays in conjugation with peptide pools representing distinct influenza virus proteins. Results. Our studies revealed that vaccination induced readily detectable CD4+ T cells specific for conserved portions of hemagglutinin (HA) and the internal viral proteins. Interestingly, expansion of HA-specific CD4+ T cells was most tightly correlated with the antibody response. Conclusions. These results indicate that CD4+ T-cell expansion may be a limiting factor in development of neutralizing antibody responses to pandemic influenza vaccines and suggest that approaches to facilitate CD4+ T-cell recruitment may increase the neutralizing antibody produced in response to vaccines against novel influenza strains.

Live attenuated H7N7 influenza vaccine primes for a vigorous antibody response to inactivated H7N7 influenza vaccine.

BACKGROUND H7 influenza viruses have emerged as potential pandemic threat. We evaluated the safety and immunogenicity of two candidate H7 pandemic live attenuated influenza vaccines (pLAIV) and their ability to prime for responses to an unadjuvanted H7 pandemic inactivated influenza vaccine (pIIV). METHODS Healthy seronegative adults received two doses of A/Netherlands/219/03 (H7N7) or one dose of A/chicken/British Columbia/CN-6/04 (H7N3) pLAIV all given as 10(7.5) 50% tissue culture infective doses (TCID50) intranasally. A subset of subjects received one 45 μg dose of H7N7 pIIV containing the A/Mallard/Netherlands/12/2000 HA intramuscularly 18-24 months after pLAIV. Viral shedding was assessed by culture and real-time polymerase chain reaction (rRT-PCR), B cell responses following pLAIV were evaluated by ELISPOT and flow cytometry. Serum antibody was assessed by hemagglutination-inhibition (HAI), microneutralization (MN) and ELISA assays after each vaccine. RESULTS Serum HAI or MN responses were not detected in any subject following one or two doses of either H7 pLAIV, although some subjects had detectable H7 specific B cells after vaccination. However, 10/13 subjects primed with two doses of H7N7 pLAIV responded to a subsequent dose of the homologous H7N7 pIIV with high titer HAI and MN antibody that cross-reacted with both North American and Eurasian lineage H7 viruses, including H7N9. In contrast, naïve subjects and recipients of a single dose of the mismatched H7N3 pLAIV did not develop HAI or MN antibody after pIIV. CONCLUSIONS While pLAIVs did not elicit detectable serum MN or HAI antibody, strain-specific pLAIV priming established long term immune memory that was cross-reactive with other H7 influenza strains. Understanding the mechanisms underlying priming by pLAIV may aid in pandemic vaccine development.

Detection of Intracellular Antigen-Specific Cytokines in Human T Cell Populations

Determination of antigen-specific cytokine responses of T lymphocytes after vaccination is made difficult by the low frequency of responder cells. In order to detect these responses, the profile of intracellular cytokines was analyzed using flow cytometry after antigenic expansion. Peripheral blood mononuclear cells were stimulated with antigens for 5 days, further expanded with interleukin (IL)-2, and then restimulated on day 10. Cytokine production was detected by intracellular staining with monoclonal antibodies after saponin-based permeabilization. Influenza expansion resulted in specific interferon-gamma (IFN-gamma) production of 6%-20%, with less IL-4 production (0%-2%). Tetanus toxoid resulted in even greater production. IL-4 and IFN-gamma were produced mainly by memory cells of the CD45RO+ phenotype. IFN-gamma production was contributed by both CD4 and CD8 populations. These methods were then applied to a clinical trial of a candidate human immunodeficiency virus type 1 vaccine. Antigen-specific increases in IFN-gamma were measured, which corresponded to antibody production, lymphoproliferation, and skin testing.

High-Affinity H7 Head and Stalk Domain–Specific Antibody Responses to an Inactivated Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine

Recent studies have shown that live attenuated influenza vaccines (LAIVs) expressing avian influenza virus hemagglutinins (HAs) prime for strong protective antibody responses to an inactivated influenza vaccine (IIV) containing the HA. To better understand this priming effect, we compared H7 HA head and stalk domain-specific B-cell responses in H7N7 LAIV-primed subjects and non-H7-primed controls after a single dose of H7N7 IIV. As previously reported, H7N7 LAIV-primed subjects but not control subjects generated strong hemagglutination-inhibiting and neutralizing antibody responses to the H7N7 IIV. Here, we found that the quantity, epitope diversity, and affinity of H7 head-specific antibodies increased rapidly in only H7N7 LAIV-primed subjects after receipt of the IIV. However, all cohorts generated a vigorous, high-affinity, stalk-specific antibody response. Consistent increases in circulating memory B-cell frequencies after receipt of the IIV reflected the specificity of high-affinity antibody production. Our findings emphasize the value of LAIVs as a vehicle for prepandemic vaccination.

Evaluation of the Safety and Immunogenicity of a Candidate Pandemic Live Attenuated Influenza Vaccine (pLAIV) Against Influenza A(H7N9).

Abstract Background. We evaluated a candidate A/Anhui/2013(H7N9) pandemic live attenuated influenza vaccine (pLAIV) in healthy adults, and assessed the ability of 1 or 2 doses to induce immune memory. Methods. Healthy subjects in 2 age groups (18–49 years and 50–70 years) with undetectable hemagglutination-inhibiting (HAI) antibody to H7N9 were enrolled. Younger subjects received either 1 or 2 intranasal doses of 107.0 fluorescent focus units of A/Anhui/1/2013 pLAIV, while older subjects received a single dose. All subjects received a single 30-µg dose of unadjuvanted, antigenically matched A/Shanghai2/2013(H7N9) pandemic inactivated influenza vaccine (pIIV) 12 weeks after their first dose of pLAIV. Results. Both vaccines were well tolerated. Serum HAI antibody responses were detected in 0 of 32 younger subjects and 1 of 17 older subjects after 1 dose of pLAIV and in 2 of 16 younger subjects after a second dose. Strong serum antibody responses were detected after a single subsequent dose of pIIV that was broadly reactive against H7 influenza viruses. Conclusions. An A(H7N9) pLAIV candidate was safe in both age groups. Priming with pLAIV resulted in responses to subsequent pIIV that exceeded those seen in naive subjects in previous reports. The A(H7N9) pLAIV induces strong immune memory that can be demonstrated by exposure to subsequent antigenic challenge. Clinical Trials Registration. NCT01995695 and NCT02274545.

Cytomegalovirus-specific IFNγ and IL-4 are produced by antigen expanded human blood lymphocytes from seropositive volunteers

The cytokine responses to cytomegalovirus (CMV) antigen in seropositive and seronegative individuals were measured using a combination of antigenic expansion and intracellular staining. Intracellular IFNgamma and IL-4 were produced in a dose-dependent manner by T cells in response to CMV only in the seropositive population. The potential for individual cells to produce both Th1 and Th2 cytokines simultaneously was clear, as IL-4 was most often produced in those cells with the highest IFNgamma production. The cytokine-specific nature of this response was demonstrated by fluorescence microscopy, which showed granular cytoplasmic staining, and at the mRNA level by ribonuclease protection assays. These methods expand our ability to evaluate the immune response to CMV, and can now be correlated to a number of clinical conditions.