In April 2009, the Centers for Disease Control and Prevention (CDC) announced the detection of a novel strain of H1N1 influenza virus in humans. The novel virus derived its genes from viruses circulating in the pig population [17
]. Due to sustained human-to-human transmission of this novel virus throughout the world, on June 11th
the World Health Organization (WHO) raised the worldwide pandemic alert level to Phase 6 [19
As H1N1pdm09 influenza enters the post-pandemic phase, health authorities around the world are reviewing the response to the pandemic to ensure this process enhances future vaccine preparations. These analyses identified several bottle-necks that require technical improvement including the need to more rapidly select optimal vaccine viruses (with high growth properties) and the need for alternative vaccine standardization technologies for potency assays and stability tests [22
Recently, several approaches have been explored as alternatives for quantification of the HA content in vaccines [23
]. To date, however, none of the newer techniques have been shown to measure the same HA antigenic form, and to be suitable for HA stability in vaccine lots. Therefore, while potentially useful, none of the newer techniques were implemented for potency determination of inactivated influenza vaccines. Furthermore, universal antibodies based on the fusion peptide in HA2 could be used in competitive ELISA, but required HA denaturation by low pH or 4–8M urea treatment [8
] and could not be used for quantitation of conformationally intact HA as required from SRID and any alternative potency methods.
Various recombinant technologies have been used to generate HA in insect and mammalian cells [28
]. Only one was approach was shown to function in SRID, but it required multiple vaccinations with alternative forms of the HA (plasmid, MVA vector, and VLP proteins) [7
]. Therefore, it was important to compare our alternative approach to generate the HA antigen for sheep immunization with the activity of sheep sera generated by the traditional method (i.e. bromelain-released HA).
Our group evaluated the possibility of producing recombinant HA in bacterial systems for the purpose of rapidly generating hyperimmune sera for the SRID-based potency assays. Within two weeks of swine-like H1N1 virus isolation from acutely infected patient in California, two HA cDNA from the A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli
under controlled redox refolding conditions that favoured proper protein folding. Surprisingly, only the recombinant HA1 (1–330) globular domain formed oligomers, showed specific binding to sialic acid receptor and agglutinated human red blood cells [9
]. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of oligomeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480) [9
In a preliminary study, rabbit immune sera generated against the purified rHA1 proteins were tested in SRID assay against H1N1pdm09 reference antigen. Good precipitation rings were observed with the sera from rHA1(1–330)-vaccinated animals but not with rHA(1–480) immune sera after second vaccination (data not shown). Based on these findings it was concluded that the presence of functional oligomers in HA preparations may be crucial for rapid generation of antibodies suitable for the SRID assay. In agreement with our previous experience with the rHA1 (1–320) produced from H5N1-A/Vietnam/1203/2004 strain [10
], we observed that the H1N1pdm09-rHA1 (1–320) protein produced in E. coli
was more stable than the rHA1 (1–330), and also agglutinated RBC better than rHA1 (1–330). The deletion of 10 amino acid sequence at the carboxy-terminus of HA1 improved HA1-oligomer stability as judged by more efficient hemeagglutination in different influenza strains including group-1 (H1N1, H5N1) and group-2 (H7N7) viruses.
In the current study, we demonstrated that the rHA1 proteins preparations contained a large fraction of oligomeric forms (rosettes of trimers) as determined by gel filtration and electron microscopy, similar to native HA spikes isolated from influenza virus. These oligomeric rHA1 were functional as measured by receptor binding assay and RBC agglutination. Importantly, the rHA1 generated high titer neutralizing antibodies in rabbits and sheep. The sera from two sheep vaccinated with the rHA1 of H1N1pdm09 according to the CBER approved protocol were found to generate good precipitation rings with H1N1pdm09 reference antigen with parallel dose-response lines when compared with the traditionally-generated sheep serum from NIBSC. The HA content in several inactivated vaccine products and multiple lots as determined in SRID assays using the rHA1-generated sheep sera were very similar to the HA content measured using the NIBSC potency reagent (i.e. values within 94%–118%). The rHA1 immunized sheep sera showed similar capacity to measure H1N1pdm09 HA content in either monovalent or trivalent vaccine formulations and were stability sensitive. Lastly, rHA1 derived from the highly pathogenic avian influenza strains A/Vietnam/1203/04 and A/Indonesia/05/05 also contained oligomers and elicited strain-specific neutralizing antibodies that behaved in the SRID assays similar to the traditional sheep sera.
These results further demonstrate that the source of immunizing antigen for generation of SRID reagent is not limited to the traditional vaccine derived bromelain-released HA. Recombinant technology could help to overcome the severe bottleneck of producing HA from influenza virus for generation of vaccine potency reagents.
In emergency situation, the HA1 of an emerging influenza strain can be chemically synthesized and cloned within one week after the HA sequence is deduced without isolating, handling, growing a pathogenic novel influenza virus or developing a reassortant vaccine strain. The purified HA1 protein can be produced within two weeks and used for sheep vaccinations.
This methodology might also be helpful to quantify and differentiate among drifted seasonal influenza strains where most of the cross-reactivity is due to the antibodies that bind to highly conserved HA2 sequences. Moreover, with ongoing efforts to develop quadrivalent seasonal influenza vaccines that includes one strain each of closely matched HA sequence but antigenically diverse Victoria and Yamagata lineage influenza B strains, it will become even more challenging to generate strain specific non-cross reactive SRID potency reagents.
However, there are several critical quality criteria that could help to determine if a given rHA1 product could be used for this purpose. Glycosylation may play more important role in the proper folding of some subtypes compared with others. In our studies to date from several group-1 influenza-A strains, one group-2 strain and two influenza-B strains, the recombinant HA proteins produced in the E. coli system, folded properly into functional oligomers with conformation that bound receptor and caused hemagglutination.
Moreover, our studies to date demonstrated that the presence of stable functional oligomers that mimic vaccine preparations is a critical requirement for generation of potency reagents for the SRID assay. RBC hemagglutination could be used as a quality control method to confirm functional oligomers in recombinant HA preparations intended for generation of potency reagents. Our study clearly demonstrated that both in the case of H1N1pdm09 and the two H5N1 AIV strains, the rHA1 elicited strain specific SRID reagents. These findings suggest that the absence of HA2 in the immunogen used for sheep vaccination had no impact on the specificity and sensitivity of the antibodies generated. Importantly, the parallel lines generated with the reference antigens in the SRID assay, suggest that the rHA1 sera recognized very similar antigenic epitopes in the vaccine products.