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Comparative Study
. 2011 Apr 5;29(16):3043-54.
doi: 10.1016/j.vaccine.2011.01.100. Epub 2011 Feb 12.

A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets

Brendan M Giles  1 , Ted M Ross
Affiliations
Comparative Study

A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets

Brendan M Giles et al. Vaccine. .

Abstract

Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naïve. Recent outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype are of particular concern because of the high mortality rate (60% case fatality rate) and novel subtype. In order to develop a vaccine that elicits broadly reactive antibody responses against emerging H5N1 isolates, we utilized a novel antigen design technique termed computationally optimized broadly reactive antigen (COBRA). The COBRA HA sequence was based upon HA amino acid sequences from clade 2 H5N1 human infections and the expressed protein retained the ability to bind the receptor, as well as mediate particle fusion. Non-infectious recombinant VLP vaccines using the COBRA HA were purified from a mammalian expression system. Mice and ferrets vaccinated with COBRA HA H5N1 VLPs had protective levels of HAI antibodies to a representative isolates from each subclade of clade 2. Furthermore, VLP vaccinated animals were completely protected from a lethal challenge of the clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. This is the first report describing the use of COBRA-based antigen design. The COBRA HA H5N1 VLP vaccine elicited broadly reactive antibodies and is an effective influenza vaccine against HPAI virus.

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Figures

Figure 1
Figure 1. COBRA HA Design
Schematic to describe how the COBRA HA molecule was designed (A). The phylogenetic tree was inferred from hemagglutinin amino acid sequences using the maximum likelihood method and clade/sub-clade groupings were identified. Primary consensus sequences were generated for each outbreak group. Secondary consensus sequences were then generated for each subclade using the primary sequences as input. The secondary consensus sequences were then aligned and the resulting consensus, designated COBRA, was generated. Phylogenetic analysis of the COBRA HA (B). The unrooted phylogenetic tree was inferred from hemagglutinin amino acid sequences from human H5N1 infections isolated from 2004 to 2009 and the clade/sub-clade groupings are indicated. The star represents the COBRA HA sequence relative to human H5N1 infections.
Figure 1
Figure 1. COBRA HA Design
Schematic to describe how the COBRA HA molecule was designed (A). The phylogenetic tree was inferred from hemagglutinin amino acid sequences using the maximum likelihood method and clade/sub-clade groupings were identified. Primary consensus sequences were generated for each outbreak group. Secondary consensus sequences were then generated for each subclade using the primary sequences as input. The secondary consensus sequences were then aligned and the resulting consensus, designated COBRA, was generated. Phylogenetic analysis of the COBRA HA (B). The unrooted phylogenetic tree was inferred from hemagglutinin amino acid sequences from human H5N1 infections isolated from 2004 to 2009 and the clade/sub-clade groupings are indicated. The star represents the COBRA HA sequence relative to human H5N1 infections.
Figure 2
Figure 2. COBRA HA Functional Characterization
COBRA HA was translated in vitro and the cell culture lysates were analyzed by SDS-PAGE (A). Lane designations: 1) H5N1 recombinant HA; 2) COBRA HA; 3) Expression vector; 4) H5N1 reassortant virus. The COBRA HA (lane 2) migrates at its expected molecular weight confirming expression of the synthetic protein. COBRA HA VLPs were prepared in various amounts, serially diluted, and incubated with 1% erythrocytes to evaluate receptor binding (B). HA titer was determined as the last well in which the RBCs remained suspended in a lattice structure. COBRA HA and control lentiviral pseudoparticles packaging a CMV-Luc gene were generated in HEK 293T cells and used to infect MDCK cells with or without trypsin (C). Particle fusion was determined by luciferace production by infected cells.
Figure 3
Figure 3. COBRA HA Mouse Dosing Immunogenicity
BALB/c mice (n=5/group) were vaccinated at 0 and 3 weeks with blood collected at 14 to 21 days after each vaccination. Vaccines were formulated at high (1.5ug HA), and low (0.03ug HA) doses; with and without Imject® alum and delivered intramuscularly. Total IgG at week 5 was determined via ELISA for each vaccine group (A and B). Values represent the geometric mean titer (±95% confidence interval) of log10 transformed endpoint titers. IgG isotypes were evaluated via ELISA for each vaccine group (C and D). Values represent the mean OD450 of a 1:200 dilution of serum. Hemagglutination inhibition (HAI) serum antibody titer for each vaccine group was determined at week 5 using representative reassortant viruses (E and F). Values represent the geometric mean titer (±95% confidence interval) of log2 transformed titers. The dotted line represents the 1:40 titer. Significant differences were determined by two-way ANOVA with Bonferroni’s post-test to evaluate differences between the vaccine formulations for each test antigen. A p value of less than 0.05 was considered significant.
Figure 3
Figure 3. COBRA HA Mouse Dosing Immunogenicity
BALB/c mice (n=5/group) were vaccinated at 0 and 3 weeks with blood collected at 14 to 21 days after each vaccination. Vaccines were formulated at high (1.5ug HA), and low (0.03ug HA) doses; with and without Imject® alum and delivered intramuscularly. Total IgG at week 5 was determined via ELISA for each vaccine group (A and B). Values represent the geometric mean titer (±95% confidence interval) of log10 transformed endpoint titers. IgG isotypes were evaluated via ELISA for each vaccine group (C and D). Values represent the mean OD450 of a 1:200 dilution of serum. Hemagglutination inhibition (HAI) serum antibody titer for each vaccine group was determined at week 5 using representative reassortant viruses (E and F). Values represent the geometric mean titer (±95% confidence interval) of log2 transformed titers. The dotted line represents the 1:40 titer. Significant differences were determined by two-way ANOVA with Bonferroni’s post-test to evaluate differences between the vaccine formulations for each test antigen. A p value of less than 0.05 was considered significant.
Figure 4
Figure 4. COBRA HA Mouse Dosing Efficacy
BALB/c mice (n=5/group) were vaccinated with COBRA HA VLPs with or without adjuvant. Mice were infected with 5×103 PFU of the highly pathogenic clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Mice were followed to monitor weight loss (A and B) and sickness (C and D). Sickness score was determined by evaluating activity (0=normal, 1=reduced, 2=severely reduced), hunched back (0=absent, 1=present) and ruffled fur (0=absent, 1=present). All mock vaccinated mice reached the experimental endpoint and required euthanasia by 6 days post infection.
Figure 5
Figure 5. Mouse Comparison Immunogenicity
BALB/c mice (n=20/group) were vaccinated at 0 and 3 weeks with blood collected at 14 to 21 days after each vaccination. Vaccines were formulated at a high dose (3ug HA) with Imject® alum and delivered intramuscularly. Total IgG at week 5 was determined via ELISA for each vaccine group (A). Values represent the geometric mean titer (±95% confidence interval) of log10 transformed endpoint titers. Hemagglutination inhibition (HAI) serum antibody titer for each vaccine group was determined at week 5 using representative reassortant viruses (B). Values represent the geometric mean titer (±95% confidence interval) of log2 transformed titers. The dotted line represents the 1:40 titer. Significant differences were determined by two-way ANOVA with Bonferroni’s post-test to evaluate differences between the vaccine formulations for each test antigen. A p value of less than 0.05 was considered significant.
Figure 6
Figure 6. Mouse Comparison Efficacy
BALB/c mice (n=20/group) were vaccinated with VLPs with adjuvant. Mice were infected with 5×103 PFU of the highly pathogenic clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Mice were followed to monitor weight loss (A) and sickness (B). Sickness score was determined by evaluating activity (0=normal, 1=reduced, 2=severely reduced), hunched back (0=absent, 1=present) and ruffled fur (0=absent, 1=present). All mock (adjuvant-only) vaccinated mice reached the experimental endpoint and required euthanasia by 6 days post infection.
Figure 7
Figure 7. Ferret Immunogenicity
Ferrets (n=9/group) were vaccinated with VLPs (15ug HA) with Imject® alum at weeks 0 and 3 and serum collected at week 5. Total IgG at week 5 was determined via ELISA for each vaccine group (A). Values represent the geometric mean titer (±95% confidence interval) of log10 transformed endpoint titers. Hemagglutination inhibition (HAI) serum antibody titer for each vaccine group was determined at week 5 using representative reassortant viruses (B). Values represent the geometric mean titer (±95% confidence interval) of log2 transformed titers. The dotted line represents the 1:40 titer. Significant differences were determined by two-way ANOVA with Bonferroni’s post-test to evaluate differences between the vaccine formulations for each test antigen. A p value of less than 0.05 was considered significant.
Figure 8
Figure 8. Ferret Efficacy
Ferrets (n=9/group) were vaccinated with VLPs formulated with adjuvant. Ferrets were challenged with 1×106 PFU of the highly pathogenic clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Animals were monitored daily for weight loss (A), survival (B), temperature (C) and clinical symptoms (D). Relative sickness scores were determined by measuring lethargy (0–3), runny nose (0–1), sneezing (0–1), loss of appetite (0–1) and diarrhea (0–1). Animals reaching experimental endpoint were euthanized according to institutional guidelines. Nasal washes were collected serially post infection and virus titers determined via plaque assay (E). Statistical significance was determined using a two-way ANOVA with Bonferroni’s post test. A p value of less than 0.05 was considered significant.
Figure 8
Figure 8. Ferret Efficacy
Ferrets (n=9/group) were vaccinated with VLPs formulated with adjuvant. Ferrets were challenged with 1×106 PFU of the highly pathogenic clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Animals were monitored daily for weight loss (A), survival (B), temperature (C) and clinical symptoms (D). Relative sickness scores were determined by measuring lethargy (0–3), runny nose (0–1), sneezing (0–1), loss of appetite (0–1) and diarrhea (0–1). Animals reaching experimental endpoint were euthanized according to institutional guidelines. Nasal washes were collected serially post infection and virus titers determined via plaque assay (E). Statistical significance was determined using a two-way ANOVA with Bonferroni’s post test. A p value of less than 0.05 was considered significant.
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