Enhancing breadth and durability of humoral immune responses in non-human primates with an adjuvanted group 1 influenza hemagglutinin stem antigen.

Seasonal influenza vaccines must be updated annually and suboptimally protect against strains mismatched to the selected vaccine strains. We previously developed a subunit vaccine antigen consisting of a stabilized trimeric influenza A group 1 hemagglutinin (H1) stem protein that elicits broadly neutralizing antibodies. Here, we further optimized the stability and manufacturability of the H1 stem antigen (H1 stem v2, also known as INFLUENZA G1 mHA) and characterized its formulation and potency with different adjuvants in vitro and in animal models. The recombinant H1 stem antigen (50 µg) was administered to influenza-naïve non-human primates either with aluminum hydroxide [Al(OH)3] + NaCl, AS01B, or SLA-LSQ formulations at week 0, 8 and 34. These SLA-LSQ formulations comprised of varying ratios of the synthetic TLR4 agonist 'second generation synthetic lipid adjuvant' (SLA) with liposomal QS-21 (LSQ). A vaccine formulation with aluminum hydroxide or SLA-LSQ (starting at a 10:25 µg ratio) induced HA-specific antibodies and breadth of neutralization against a panel of influenza A group 1 pseudoviruses, comparable with vaccine formulated with AS01B, four weeks after the second immunization. A formulation with SLA-LSQ in a 5:2 µg ratio contained larger fused or aggregated liposomes and induced significantly lower humoral responses. Broadly HA stem-binding antibodies were detectable for the entire period after the second vaccine dose up to week 34, after which they were boosted by a third vaccine dose. These findings inform about potential adjuvant formulations in clinical trials with an H1 stem-based vaccine candidate.

Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions.

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit ß-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Immunogenicity and efficacy of one and two doses of Ad26.COV2.S COVID vaccine in adult and aged NHP.

Safe and effective coronavirus disease-19 (COVID-19) vaccines are urgently needed to control the ongoing pandemic. While single-dose vaccine regimens would provide multiple advantages, two doses may improve the magnitude and durability of immunity and protective efficacy. We assessed one- and two-dose regimens of the Ad26.COV2.S vaccine candidate in adult and aged nonhuman primates (NHPs). A two-dose Ad26.COV2.S regimen induced higher peak binding and neutralizing antibody responses compared with a single dose. In one-dose regimens, neutralizing antibody responses were stable for at least 14 wk, providing an early indication of durability. Ad26.COV2.S induced humoral immunity and T helper cell (Th cell) 1-skewed cellular responses in aged NHPs that were comparable to those in adult animals. Aged Ad26.COV2.S-vaccinated animals challenged 3 mo after dose 1 with a SARS-CoV-2 spike G614 variant showed near complete lower and substantial upper respiratory tract protection for both regimens. Neutralization of variants of concern by NHP sera was reduced for B.1.351 lineages while maintained for the B.1.1.7 lineage independent of Ad26.COV2.S vaccine regimen.

Rescue of recent virulent and avirulent field strains of bluetongue virus by reverse genetics.

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of 'synthetic BTV', respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these 'synthetic BTVs' could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

Bluetongue virus serotype 6 in Europe in 2008-Emergence and disappearance of an unexpected non-virulent BTV.

Bluetongue viruses (BTVs) could invade N-W Europe similar to BTV serotype 8 (BTV8/net06), since the source and route of introduction of this virus has not been solved. Therefore, the Dutch survey for Bluetongue by PCR testing was extended by further analysis of PCR positives to identify the involved BTV. In late August 2008, BTV was reported with 12 nucleotide differences in the S10 amplicon (S10 genotyping). This virus was identified as serotype 6, here named BTV6/net08. Promptly, serotype specific real-time PCR tests were developed for serotypes 1, 6, and 8 (S2 genotyping). Agreement was found between results by S10- and S2 genotyping. Further, BTV1 was identified by both S10- and S2 genotyping in one imported animal. After initial discovery of BTV6 in the Netherlands, animals from 18 holdings tested PCR positive for BTV6/net08 in 2008. Remarkably only one or two PCR positive animals per holding were found. Serum neutralization tests did not result in the discovery of more BTV6 infected animals. Retrospective studies indicated no evidence for infections by BTV6/net08 prior to the first discovery. Experimental infections with BTV6/net08 did not cause clinical disease in sheep, calves and cattle, except for a very short fever in some animals. This clearly showed that the vaccine-related BTV6/net08 is not virulent. BTV6/net08 was not found by passive and active surveys in the years after its discovery. Apparently, BTV6/net08 was not efficiently transmitted by endemic species of Culicoides in N-W Europe, and disappeared without the need of any control measure.

Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments.

Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in The Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus Orbivirus for which reverse genetics has not been developed yet.