Development of Cross-Reactive Live Attenuated Influenza Vaccine Candidates against Both Lineages of Influenza B Virus

Abstract

Background: Influenza viruses continue to cause a significant social and economic burden globally. Vaccination is recognized as the most effective measure to control influenza. Live attenuated influenza vaccines (LAIVs) are an effective means of preventing influenza, especially among children. A reverse genetics (RG) system is required to rapidly update the antigenic composition of vaccines, as well as to design LAIVs with a broader spectrum of protection. Such a system has been developed for the Russian LAIVs only for type A strains, but not for influenza B viruses (IBV).

Methods: All genes of the B/USSR/60/69 master donor virus (B60) were cloned into RG plasmids, and the engineered B60, as well as a panel of IBV LAIV reassortants were rescued from plasmid DNAs encoding all viral genes. The engineered viruses were evaluated in vitro and in a mouse model.

Results: The B60 RG system was successfully developed, which made it possible to rescue LAIV reassortants with the desired antigenic composition, including hybrid strains with hemagglutinin and neuraminidase genes belonging to the viruses from different IBV lineages. The LAIV candidate carrying the HA of the B/Victoria-lineage virus and NA from the B/Yamagata-lineage virus demonstrated optimal characteristics in terms of safety, immunogenicity and cross-protection, prompting its further assessment as a broadly protective component of trivalent LAIV.

Conclusions: The new RG system for B60 MDV allowed the rapid generation of type B LAIV reassortants with desired genome compositions. The generation of hybrid LAIV reassortants with HA and NA genes belonging to the opposite IBV lineages is a promising approach for the development of IBV vaccines with broad cross-protection.

Keywords: cross-protection; influenza B virus; live attenuated influenza vaccine; recombinant influenza virus; reverse genetics; viral immunity.

Figure 1

Growth characteristics of recombinant LAIV viruses in MDCK cells. Confluent monolayers of MDCK cells were inoculated at an MOI of 0.001 with either the MDV or 6 + 2 (RG and non-RG) and incubated at 33 °C. Culture supernatants were collected at 0, 24, 48, 72 and 96 hpi, and viral titers were quantified by TCID₅₀ assays. Dotted line represents the limit of virus detection in the TCID₅₀ assay.

Figure 2

Replication of the studied influenza B viruses in mouse respiratory tissues. Viruses were administered i.n. to groups of mice (n = 4) at a dose of 10⁶ EID₅₀. Lungs and nasal turbinates (NT) were collected on Day 3 p.i. and viral titers were determined by the titration of tissue homogenates in eggs. Data were compared using two-way ANOVA with Tukey’s post-hoc multiple analyses test. ** p < 0.01, *** p < 0.001. Dotted line represents the limit of virus detection in the EID₅₀ assay.

Figure 3

Antibody immune responses to the RG and non-RG influenza B viruses in mice after two immunizations. (A) Neutralizing antibody titers to the Ph-wt virus as measured by MN assay. (B) Binding levels of serum IgG antibodies to the purified Ph-wt whole virus as measured by ELISA. (C) Endpoint serum IgG antibody titers to the Ph-wt virus determined by ELISA. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. **—p < 0.01; ***—p < 0.001; ****—p < 0.0001.

Figure 4

Protective activity of RG and non-RG influenza B viruses in mice. Immunized mice were challenged with B/Phuket/3037/2013 virus, and viral titers were determined in lungs and nasal turbinates on Day 3 post challenge. Data were analyzed by two-way ANOVA with Tukey’s post-hoc multiple analyses test. **—p < 0.01; ***—p < 0.001; ****—p < 0.0001. Dotted line represents the limit of virus detection in the TCID₅₀ assay.

Figure 5

Hybrid (6 + 1 + 1) and control (6 + 2) recombinant viruses rescued for this experiment.

Figure 6

Safety and immunogenicity of viruses. Virus replication and tissue tropism of the 6 + 2 or 6 + 1 + 1 viruses in the respiratory tracts of mice. At 3 dpi, four animals from each group were euthanized, and virus titers in the upper respiratory tracts (nasal turbinates) or lower respiratory tracts (lungs) of the mice were determined by limiting dilutions in eggs. Data were analyzed by two-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; ****—p < 0.0001. Dotted line represents the limit of virus detection in the EID₅₀ assay.

Figure 7

Vaccination with 6 + 1 + 1 constructs induces antibody responses against diverse IBVs. Serum IgG responses to (A) Br-wt and (B) Ph-wt, as measured by HAI assay and by ELISA. Left panel shows the results of HAI assay (mean ± SD of HAI titers). Middle panel shows the mean ± SD OD₄₅₀ values for serum dilutions in each group in ELISA. Right panel shows the mean + SD of area under the curve (AUC) of OD₄₅₀ values as a readout of ELISA. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ***—p < 0.001; ****—p < 0.0001.

Figure 7

Vaccination with 6 + 1 + 1 constructs induces antibody responses against diverse IBVs. Serum IgG responses to (A) Br-wt and (B) Ph-wt, as measured by HAI assay and by ELISA. Left panel shows the results of HAI assay (mean ± SD of HAI titers). Middle panel shows the mean ± SD OD₄₅₀ values for serum dilutions in each group in ELISA. Right panel shows the mean + SD of area under the curve (AUC) of OD₄₅₀ values as a readout of ELISA. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ***—p < 0.001; ****—p < 0.0001.

Figure 8

NA-inhibiting (NI) antibody titers in serum. Serial dilutions of 7 + 1 Len/17-based diagnostic strain with NA of Br-wt (A) or Ph-wt (B) were incubated for 1 h at 37 °C in fetuin-coated plates and the reactivity with PNA-HRPO was determined as described. The dilution of H2Br selected for use in the ELLA was 1:640 and the dilution of H2Ph selected was 1:640 because these dilutions resulted in approximately 90% of the maximum optical density and were within the linear range. Data were compared with one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ****—p < 0.0001.

Figure 9

Weight loss and survival of vaccinated groups challenged with (A) 6 logEID₅₀ or (B) 5 logEID₅₀ of mouse-adapted B/Malaysia/2506/2004 virus in mice. Left panel shows dynamics of body weight over the challenge phase. Right panel shows survival rates among all mice used in the experiment.

Figure 10

Protective activity of 6 + 2 and 6 + 1 + 1 LAIV viruses in mice. Immunized mice were challenged with B/Brisbane/60/2008 (left panel) and B/Phuket/3037/2013 virus (right panel), and viral titers were determined in lungs and nasal turbinates on Day 3 post challenge. Data were compared with two-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ****—p < 0.0001. Dotted line represents the limit of virus detection in the EID₅₀ assay.

Figure 11

Indirect protective effect of the sera from mice immunized with LAIV prototypes generated in this study. Pooled sera collected from immunized mice at Day 43 were mixed with (A) Br-wt, (B) Ph-wt, or (C) Ma-wt virus at a dose of 3 MLD₅₀ and inoculated intranasally to naïve C57BL/6J mice (n = 5–6). Survival and weight loss were monitored for 14 days post-challenge. Data were analyzed by one-way ANOVA with Tukey’s post-hoc multiple analyses test. *—p < 0.05; **—p < 0.01; ***—p < 0.001; ****—p < 0.0001.