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. 2024 May 14;12(5):537.
doi: 10.3390/vaccines12050537.

Very Broadly Effective Hemagglutinin-Directed Influenza Vaccines with Anti-Herpetic Activity

David C Bloom  1 , Cameron Lilly  1 , William Canty  1 , Nuria Vilaboa  2   3 , Richard Voellmy  4
Affiliations

Very Broadly Effective Hemagglutinin-Directed Influenza Vaccines with Anti-Herpetic Activity

David C Bloom et al. Vaccines (Basel). .

Abstract

A universal vaccine that generally prevents influenza virus infection and/or illness remains elusive. We have been exploring a novel approach to vaccination involving replication-competent controlled herpesviruses (RCCVs) that can be deliberately activated to replicate efficiently but only transiently in an administration site in the skin of a subject. The RCCVs are derived from a virulent wild-type herpesvirus strain that has been engineered to contain a heat shock promoter-based gene switch that controls the expression of, typically, two replication-essential viral genes. Additional safety against inadvertent replication is provided by an appropriate secondary mechanism. Our first-generation RCCVs can be activated at the administration site by a mild local heat treatment in the presence of an antiprogestin. Here, we report that epidermal vaccination with such RCCVs expressing a hemagglutinin or neuraminidase of an H1N1 influenza virus strain protected mice against lethal challenges by H1N1 virus strains representing 75 years of evolution. Moreover, immunization with an RCCV expressing a subtype H1 hemagglutinin afforded full protection against a lethal challenge by an H3N2 influenza strain, and an RCCV expressing a subtype H3 hemagglutinin protected against a lethal challenge by an H1N1 strain. Vaccinated animals continued to gain weight normally after the challenge. Protective effects were even observed in a lethal influenza B virus challenge. The RCCV-based vaccines induced robust titers of in-group, cross-group and even cross-type neutralizing antibodies. Passive immunization suggested that observed vaccine effects were at least partially antibody-mediated. In summary, RCCVs expressing a hemagglutinin induce robust and very broad cross-protective immunity against influenza.

Keywords: broad protection; conditionally replicating; influenza; regulated; replication-competent; universal flu vaccine; vaccine; vectored vaccine.

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Conflict of interest statement

Richard Voellmy is the founder of HSF Pharmaceuticals SA. The company had no role in data collection and interpretation. HSF Pharmaceuticals SA has property interests in RCCV technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Two-component gene switch, schematic representation of the structures of RCCVs, regulation of RCCV replication and expression of HAs from RCCVs. (a) Dually responsive gene switch in HSV-GS recombinants: a promoter assembly comprising an HSP70B promoter (HSP70B) and a GAL4-responsive promoter (GAL4) controls a gene for antiprogestin (AP)-activated transactivator GLP65. The replication-essential ICP4 and ICP8 genes are controlled by GAL4 promoters. Heat treatment of a cell infected with an HSV-GS recombinant transiently activates the cellular heat shock factor (HSF1) that then transactivates the GLP65 gene. Newly synthesized, inactive GLP65 molecules are activated when bound by an AP. Activated GLP65 transactivates the GAL4 promoter-controlled ICP4 and ICP8 genes as well as its own gene. (b) Diagram of RCCV HSV-GS3. The recombinant comprises, inserted in the UL43/44 intergenic region of HSV-1 wild-type strain 17syn+, transactivator gene GLP65, which is functionally linked to an HSP70B/GAL4 (GAL4-responsive promoter) promoter assembly. GLP65 is a chimeric transcription factor comprising a yeast-derived GAL4 DNA-binding domain, an antiprogestin-binding domain derived from the ligand-binding domain of a human progesterone receptor and an activation domain from the human P65 protein. Antiprogestin-activated GLP65 transactivates GAL4-responsive promoters. The native promoters of the replication-essential genes encoding ICP4 (both copies) and ICP8 in HSV-1 strain 17syn+ were replaced with GAL4-responsive promoters. (c) Diagram of RCCVs HSV-GS19 and HSV-GS26. The RCCVs are derived from HSV-GS3 and additionally comprise a CMV IE promoter-driven gene encoding the HA of influenza virus strain A/California/07/2009 (CA09) (HSV-GS19) or A/Hong Kong/4801/2014 (HK14) (HSV-GS26) inserted in the UL37/38 intergenic region. TRL, TRS: long and short terminal repeats; UL, US: long and short unique regions; IRL, IRS: long and short internal repeats. (d,e) Gene switch-controlled replication of RCCVs HSV-GS19 and HSV-GS26. Single-step growth experiments with HSV-GS19 (d) and HSV-GS26 (e) were carried out in Vero cells. Heat: cultures were exposed to 43.5 °C for 30 min immediately after infection (i.e., immediately after removal of the viral inoculum); Uli: 10 nM ulipristal was added to the medium at the time of infection. Mean values of the results of 3 individual assays are presented. The values are expressed as log10 total plaque-forming units (PFU). * p ≤ 0.05 (compared with cells treated with heat and ulipristal at 12 h or 24 h). (f) Regulated expression of HAs from RCCVs HSV-GS19 and HSV-GS26. Adult BALB/c mice (groups of 3 mice) were inoculated on their rear footpads either with saline (mock) or with HSV-GS3, HSV-GS19 or HSV-GS26 (all at 5 × 104 PFU). Activation was by a 10 min immersion of the hindlegs in a 45 °C water bath 3 h after inoculation in the presence of ulipristal (50 µg/kg; administered IP at the time of virus administration). Tissue samples were harvested from mouse feet 24 h later, and protein homogenates were prepared and analyzed by HA-specific ELISA. The data represent the mean values of the chromogenic signals of the samples minus the mean value of the negative control relative to the mean value of the negative control.
Figure 2
Figure 2
Homologous influenza virus challenges. Groups (n = 10) of adult BALB/c mice were inoculated on the slightly abraded plantar surfaces of their rear feet with 2.5 × 105 PFU of RCCV HSV-GS19 or HSV-GS26 or vehicle. RCCV replication was activated by a local heat treatment in the systemic presence of ulipristal. Heat treatment for 10 min at 44.5 °C was performed by immersion of hindlimbs in a temperature-controlled water bath 3 h after virus administration. Ulipristal (50 µg/kg) in DMSO was administered IP at the time of virus inoculation. Inoculations (with the same RCCVs and at the same doses of RCCVs) and activation treatments were repeated 3 wk later. After a further 3 wk, all mice were challenged intranasally with a lethal dose of the homologous influenza virus strain. Animals were observed daily, and weights were recorded. Left graphs: survival (≤20% weight loss) after challenge; center graphs: averaged relative weights of surviving animals after challenge. Weights are relative to weights on the day of challenge. Relative values and standard deviations are shown. p ≤ 0.05 (compared with mock-immunized animals (*) or to animals immunized with not-activated HSV-GS19 (#, &)); right graphs: relative weights after challenge of all animals in the groups vaccinated with activated RCCV. Weights are relative to weights on the day of challenge. (a) Mice immunized twice with activated or not-activated RCCV HSV-GS19 (expressing the HA of influenza virus strain A/California/07/2009 (CA09)) or vehicle (mock) and challenged with a lethal dose of influenza virus strain CA09. (b) Mice immunized twice with activated RCCV HSV-GS26 (expressing the HA of influenza virus strain A/Hong Kong/4801/2014 (HK14)) or vehicle (mock) and challenged with a lethal dose of influenza virus strain HK14.
Figure 3
Figure 3
Heterologous (intra-subtypic) influenza virus challenges. (a) Diagram illustrating the phylogenetic relationships between the HA expressed by RCCV-GS19 and the HAs of the challenge virus strains employed (adapted from ref. [44]). (b–d) Groups (n = 10) of adult BALB/c mice (or DBA/2 mice for challenges with strain A/Solomon Islands/3/2006 (SI06)) were immunized twice with 2.5 × 105 PFU/mouse of not-activated or activated RCCV HSV-GS19, activated recombinants HSV-GS21 or HSV-GS25, or vehicle as detailed in Figure 2. RCCV HSV-GS19 expresses a full-length HA of H1N1 strain A/California/07/2009 (CA09), RCCV HSV-GS21, a full-length NA of strain CA09, and RCCV HSV-GS25, a stem region fragment containing HA sequences of strain A/Puerto Rico/8/1934 (PR34). Three wk after the second immunization, the mice were challenged intranasally with lethal doses of H1N1 influenza virus strains A/Fort Monmouth/1/1947 (FM47) (b), SI06 (c) or PR34 (d). The animals were observed daily, and their weights were recorded. Left graphs: survival (≤20% weight loss) after challenge; center graphs: averaged relative weights of surviving animals after challenge. Weights are relative to weights on the day of challenge. Relative values (down to the nadir in the case of control groups comprising surviving animals) and standard deviations are shown; right graphs: relative weights after challenge of all animals in the groups vaccinated with an activated RCCV. Weights are relative to weights on the day of challenge. (e,f) Similar experiment in which groups (n = 10) of adult BALB/c (e) or DBA/2 (f) mice were immunized twice with 5 × 104 PFU/mouse of not-activated or activated HSV-GS19 or a combination of activated HSV-GS19 and HSV-GS21 (5 × 104 PFU each), or vehicle and challenged with lethal doses of H1N1 strains FM47 (e) or SI06 (f). (g) Groups (n = 10) of adult mice were immunized twice with 1 × 104 PFU/mouse of activated RCCVs HSV-GS19 or HSV-GS25, or vehicle and were challenged with strain FM47. Center graphs: p ≤ 0.05 (comparing the mock-immunized group to any RCCV-immunized group shown in the graph (*), to the activated HSV-GS19 or the HSV-GS21 group ($), to the activated HSV-GS19 or the HSV-GS25 group (&), or to the activated HSV-GS19 or the HSV-GS19/HSV-GS21 group (†), comparing the not-activated HSV-GS19 group with the activated HSV-GS19 or the HSV-GS19/HSV-GS21 group (Ω), comparing the activated HSV-GS19 group with the HSV-GS19/HSV-GS21 group (#) or the activated HSV-GS25 group (Φ) or comparing the activated HSV-GS21 group with the activated HSV-GS19 group (Ç)).
Figure 4
Figure 4
Heterologous (intra-subtypic), cross-group and cross-type influenza virus challenges, and an HSV-1 challenge. Groups (n = 10 or 30 in (f)) of adult BALB/c mice were immunized twice with 2.5 × 105 PFU/mouse of the indicated activated RCCVs or vehicle and were challenged with a lethal dose of the indicated influenza virus strains as described in Figure 2. (a) Mice immunized with RCCV HSV-GS27 (expressing the HA of H3N2 influenza virus strain A/Perth/16/2009) or vehicle (mock) and challenged with H3N2 influenza virus strain A/Hong Kong/4801/2014 (HK14). (b) Mice immunized with RCCV HSV-GS19 (expressing the HA of H1N1 influenza virus strain A/California/07/2009 (CA09)) or vehicle (mock) and challenged with H3N2 influenza virus strain A/Hong Kong/4801/2014 (HK14). (c) Mice immunized with RCCV HSV-GS26 (expressing the HA of H3N2 influenza virus strain A/Hong Kong/4801/2014 (HK14)) or vehicle (mock) and challenged with H1N1 influenza virus strain A/California/07/2009 (CA09). (d) Mice immunized with RCCV HSV-GS26 (expressing the HA of H3N2 influenza virus strain A/Hong Kong/4801/2014 (HK14)) or vehicle (mock) and challenged with influenza B virus strain B/Brisbane/60/2008 (BR08). (e) As (d), except that animals were immunized with a combination of HSV-GS19 and HSV-GS26 (both at 2.5 × 105 PFU/mouse). Left graphs: survival (≤20% weight loss) after challenge; center graphs: averaged relative weights of surviving animals after challenge. Weights are relative to weights on the day of challenge. Relative values and standard deviations are shown. * p ≤ 0.05 (compared with groups immunized with the indicated RCCV); right graphs: relative weights after challenge of all animals in the RCCV-vaccinated groups. Weights are relative to weights on the day of challenge. (f) Lung titers of challenge virus HK14. The results are presented as TCID50 values per g of tissue. Limit of detection: 210 TCID50/g tissue. ND: not detected. * p ≤ 0.05 (compared with groups immunized with RCCV). The experiment is described in the results. (g) HSV-1 challenge. A group (n = 10) of adult BALB/c mice that had previously been immunized twice with 2.5 × 105 PFU/mouse of RCCV HSV-GS19 and challenged with a lethal dose of influenza virus strain FM47 and an age-matched control group were challenged (3 wk after the earlier challenge) with a lethal dose of HSV-1 wild-type strain 17syn+. Animals were observed daily for 3 wk, and survival was recorded.
Figure 5
Figure 5
Neutralizing antibodies, passive immunization and a challenge experiment with JHT mice. (a) Neutralizing antibody responses. Groups of adult BALB/c mice (n = 10) were pre-bled and bled again 3 wk after the first immunization with the indicated RCCV or vehicle (mock) and 3 wk after the second immunization. Sera were prepared for each animal in the respective groups and analyzed for neutralizing antibodies against the indicated influenza virus strains using the microneutralization assay described in Section 2. Data are presented as neutralization ID50 titers (reciprocal dilutions where infection was reduced by 50% relative to normal serum expressed as geometric mean ID50). Lines indicate mean ± standard deviation. ND: not detected (below the limit of detection). p ≤ 0.05 (compared with the mock group (*) or the not-activated HSV-GS19 group (#)). (b) Passive immunization. Forty adult BALB/c mice were immunized twice with 2.5 × 105 PFU/mouse of RCCV HSV-GS19. Three wk after the second immunization, total blood volumes were collected and combined and serum was prepared. Undiluted or 10-fold diluted serum (1 mL) was administered IP to groups of naive adult BALB/c mice (n = 10). Twenty-four h later, all animals were challenged with a lethal dose of either influenza virus strain A/California/07/2009 (CA09) or A/Hong Kong/4801/2014 (HK14). Left graphs: survival (≤20% weight loss) after challenge; center graphs: averaged relative weights of surviving animals after challenge. Weights are relative to weights on the day of challenge. Relative values and standard deviations are shown. p ≤ 0.05 (compared with the mock group (*) or to the group of animals administered 10-fold diluted serum (#)); right graphs: relative weights after challenge of all animals of the group that had received undiluted serum. Weights are relative to weights on the day of challenge. (c) Challenge experiment employing JHT mice. Groups (n = 10) of adult JHT mice were immunized twice with 2.5 × 105 PFU/mouse of activated RCCV HSV-GS19 or vehicle and were challenged with a lethal dose of influenza virus strain HK14, as described in Figure 2. * p ≤ 0.05 (compared with the mock group). See under (b) for details of presentation.
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