Immunogenicity and protective efficacy of a co-formulated two-in-one inactivated whole virus particle COVID-19/influenza vaccine.

Due to the synchronous circulation of seasonal influenza viruses and severe acute respiratory coronavirus 2 (SARS-CoV-2) which causes coronavirus disease 2019 (COVID-19), there is need for routine vaccination for both COVID-19 and influenza to reduce disease severity. Here, we prepared individual WPVs composed of formalin-inactivated SARS-CoV-2 WK 521 (Ancestral strain; Co WPV) or influenza virus [A/California/07/2009 (X-179A) (H1N1) pdm; Flu WPV] to produce a two-in-one Co/Flu WPV. Serum analysis from vaccinated mice revealed that a single dose of Co/Flu WPV induced antigen-specific neutralizing antibodies against both viruses, similar to those induced by either type of WPV alone. Following infection with either virus, mice vaccinated with Co/Flu WPV showed no weight loss, reduced pneumonia and viral titers in the lung, and lower gene expression of proinflammatory cytokines, as observed with individual WPV-vaccinated. Furthermore, a pentavalent vaccine (Co/qFlu WPV) comprising of Co WPV and quadrivalent influenza vaccine (qFlu WPV) was immunogenic and protected animals from severe COVID-19. These results suggest that a single dose of the two-in-one WPV provides efficient protection against SARS-CoV-2 and influenza virus infections with no evidence of vaccine interference in mice. We propose that concomitant vaccination with the two-in-one WPV can be useful for controlling both diseases.

Assessing the pyrogenicity of whole influenza virus particle vaccine in cynomolgus macaques.

Among inactivated influenza vaccines, the whole virus particle vaccine (WPV) elicits superior priming responses to split virus vaccine (SV) in efficiently inducing humoral and cellular immunity. However, there is concern for undesired adverse events such as fever for WPV due to its potent immunogenicity. Therefore, this study investigated the febrile response induced by subcutaneous injection with quadrivalent inactivated influenza vaccines of good manufacturing grade for pharmaceutical or investigational products in cynomolgus macaques. Body temperature was increased by 1 °C-2 °C for 6-12 h after WPV administration at the first vaccination but not at the second shot, whereas SV did not affect body temperature at both points. Given the potent priming ability of WPV, WPV-induced fever may be attributed to immune responses that uniquely occur during priming. Since WPV-induced fever was blunted by pretreatment with indomethacin (a cyclooxygenase inhibitor), the febrile response by WPV is considered to depend on the increase in prostaglandins synthesized by cyclooxygenase. In addition, WPV, but not SV, induced the elevation of type I interferons and monocyte chemotactic protein 1 in the plasma; these factors may be responsible for pyrogenicity caused by WPV, as they can increase prostaglandins in the brain. Notably, sufficient antibody responses were acquired by half the amount of WPV without causing fever, suggesting that excessive immune responses to trigger the febrile response is not required for acquired immunity induction. Thus, we propose that WPV with a reduced antigen dose should be evaluated for potential clinical usage, especially in naïve populations.

Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques.

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naïve individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naïve cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage FcγR2a and FcγR3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-γ-producing CD4+ T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naïve individuals and also in the event of a pandemic outbreak.

Potent priming by inactivated whole influenza virus particle vaccines is linked to viral RNA uptake into antigen presenting cells.

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8+ and CD4+ T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.

Clinical phase II and III studies of an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform.

BACKGROUND: We have developed an AS03-adjuvanted H5N1 influenza vaccine produced in an EB66® cell culture platform (KD-295). OBJECTIVES: In accordance with Japanese guidelines for development of pandemic prototype vaccines, the phase II study was conducted in a double-blind, randomized, parallel-group comparison study and the phase III study was conducted in an open-label, non-randomized, uncontrolled study. METHODS: Healthy adult volunteers aged 20 - 64 years enrolled in the phase II and III studies (N = 248 and N = 369) received KD-295 intramuscularly twice with a 21-day interval. After administration, immune response and adverse events were evaluated. In the phase II study, four different vaccine formulations were compared: MA (3.75 µg hemagglutinin [HA] antigen + AS03 adjuvant system), MB (3.75 µg HA + 1/2AS03), HA (7.5 µg HA + AS03), and HB (7.5 µg HA + 1/2AS03). In the phase III study, the MA formulation was further evaluated. RESULTS: In the phase II study, all four vaccine formulations were well-tolerated and no SAE related to vaccination were observed. The MA formulation was slightly more immunogenic and less reactogenic among the vaccine formulations. Therefore, the MA formulation was selected for the phase III study, and it was well-tolerated and no serious adverse drug reactions were observed. The vaccine fulfilled the three immunogenicity criteria described in the Japanese guidelines. CONCLUSIONS: These data indicate that the MA formulation of KD-295 was well-tolerated and highly immunogenic and it can be considered a useful pandemic and pre-pandemic influenza vaccine.

Inactivated whole virus particle vaccine with potent immunogenicity and limited IL-6 induction is ideal for influenza.

In contrast to current ether- or detergent-disrupted "split" vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8+ T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.

Treatment of breast cancer cells with proteasome inhibitor lactacystin increases the level of sensitivity to cell death induced by human immunodeficiency virus type 1.

Upon binding to CD4, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 undergoes conformational changes that facilitate subsequent interactions with the chemokine coreceptor CXCR4 on the T cells. Our previous study showed that HIV-1 induces breast cancer cell death through gp120-CXCR4 interaction without CD4-induced conformational change of gp120. To characterize the structural properties of CXCR4 on breast cancer cells, the structural differences in CXCR4 between breast cancer cell lines and T cells were investigated. Immunoblots of whole cell lysates from breast cancer cell and T cell lines demonstrated that the predominant forms of CXCR4 on the breast cancer cell lines and T cell lines were three species (45, 61, 100 kDa) and one species (45 kDa), respectively. Cell surface biotin labeling revealed that the 100-kDa polyubiquitinated form of CXCR4 is specifically expressed on the surface of breast cancer cell line DU4475 but not T cell line Molt4#8. The treatment of breast cancer cell lines MDA-MB231 and DU4475 with proteasome inhibitor lactacystin leads to increased surface expression of the 100-kDa polyubiquitinated form of CXCR4 and increases the level of sensitivity to cell death induced by HIV-1. These data suggest that the 100-kDa polyubiquitinated form of CXCR4 plays an important role as a trigger for gp120-induced breast cancer cell death.

Human immunodeficiency virus-induced apoptosis of human breast cancer cells via CXCR4 is mediated by the viral envelope protein but does not require CD4.

HIV-1 infection results in an increased risk of malignancy as well as immune suppression. However, analyses of cancer incidence in chronically immunosuppressed transplant recipients and HIV-infected person have demonstrated an unexpected low incidence of certain types of cancer, such as breast cancers, and the mechanism behind this remains unclarified. In this study, we show that most breast cancer cell lines express CXCR4 but are not susceptible to HIV-1 infection. The apoptosis of breast cancer cells is induced by HIV-1 in a viral-dose- and time-dependent manner without productive infection. The apoptosis is induced by R5X4 and X4 HIV-1 but not by R5 HIV-1, and is inhibited by an anti-CXCR4 antibody, an anti-gp120 antibody, AMD3100, or pertussis toxin. The apoptosis is mediated via CXCR4 in breast cancer cells that exhibit conformational heterogeneity in comparison with CXCR4 in T-cells. Furthermore, the gp120 mutant (E370R) with a low CD4 binding ability can specifically induce apoptosis in breast cancer cells but not in T-cells. Taken together, these results indicate that HIV-1 and gp120 can induce breast cancer cell apoptosis through gp120-CXCR4 interaction without a CD4-induced conformational change of gp120, and may lead to a novel HIV-1-based therapy for breast cancer.

Effects of immunization with CCR5-based cycloimmunogen on simian/HIVSF162P3 challenge.

A synthetic cycloimmunogen targeting the HIV-1 coreceptor CCR5 was evaluated for its capacity to induce CCR5-specific Abs with anti-HIV-1 activity in cynomolgus macaques. The cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of human CCR5 was chemically prepared, in which the Gly-Glu dipeptide links the amino and carboxy termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (Arg168 to Cys178) of extracellular loop-2 in CCR5. The immunization of cynomolgus macaques with the cDDR5-conjugated multiple-Ag peptide (cDDR5-MAP) induced anti-cDDR5 serum production for approximately 15 wk after the third immunization. The antisera raised against cDDR5-MAP reacted with both human and macaque CCR5s, and potently suppressed infection by the R5 HIV-1 laboratory isolate (HIV JRFL), R5 HIV-1 primary isolates (clade A:HIV 93RW004 and clade C:HIV MJ4), and a pathogenic simian/HIV (SHIV SF162P3) bulk isolate in vitro. To examine the prophylactic efficacy of anti-CCR5 serum Ab for acute HIV-1 infection, cynomolgus macaques were challenged with SHIV SF162P3. The cDDR5-MAP immunization attenuated the acute phase of SHIV SF162P3 replication. The geometric mean plasma viral load in the vaccinated macaques was 217.10 times lower than that of the control macaques at 1 wk postchallenge. Taken together, these results suggest that cDDR5-MAP immunization is an effective prophylactic vaccine strategy that suppresses and delays viral propagation during the initial HIV-1 transmission for the containment of HIV-1 replication subsequent to infection.

A novel cyclic peptide immunization strategy for preventing HIV-1/AIDS infection and progression.

A novel synthetic peptide immunogen targeting the human immunodeficiency virus type-1 (HIV-1) coreceptor CXCR4 was evaluated for its capacity to induce CXCR4-specific antibodies with anti-HIV-1 activity in BALB/c mice and cynomolgus monkeys. A cyclic closed-chain dodecapeptide mimicking the conformation-specific domain of CXCR4 (cDDX4) was prepared in which Gly-Asp, as the dipeptide forming a spacer arm, links the amino and carboxyl termini of the decapeptidyl linear chain (linear DDX4, Asn176 to Ile185) derived from the undecapeptidyl arch (UPA; Asn176 to Cys186) of extracellular loop 2 (ECL-2) in CXCR4. Immunization of BALB/c mice with cDDX4 conjugated with a multiple-antigen peptide (cDDX4-MAP) induced conformational epitope-specific antibodies, and monoclonal antibody IA2-F9 reacted with cDDX4, but not with linear DDX4, as determined by real-time biomolecular interaction analysis using surface plasmon resonance. The antibody also reacted with cells expressing CXCR4 but not with cells expressing the other HIV coreceptor, CCR5. Furthermore, the antibody inhibited the replication of HIV-1 X4 virus (using CXCR4), as shown by an infection assay using both MAGIC-5 cells and MT4 cells, but not that of HIV-1 R5 virus (using CCR5). The antibody weakly interfered with chemotaxis induced by stromal cell-derived factor-1 alpha in THP-1 cells or moderately inhibited the chemotaxis of Molt4#8 cells under the same conditions. In addition, immunization of cynomolgus monkeys also induced cDDX4-specific antibodies with anti-HIV activity. Taken together, these results indicate that cDDX4 conjugated with a multi-antigen peptide induces the conformational epitope-specific antibodies to the undecapeptidyl arch of CXCR4 may be a novel candidate immunogen for preventing disease progression in HIV-1-infected individuals.