A Vaccine Based on the A/ASIA/G-VII Lineage of Foot-and-Mouth Disease Virus Offers Low Levels of Protection against Circulating Viruses from the A/ASIA/Iran-05 lineage

The recent emergence and circulation of the A/ASIA/G-VII (A/G-VII) lineage of foot-and-mouth disease virus (FMDV) in the Middle East has resulted in the development of homologous vaccines to ensure susceptible animals are sufficiently protected against clinical disease. However, a second serotype A lineage called A/ASIA/Iran-05 (A/IRN/05) continues to circulate in the region and it is therefore imperative to ensure vaccine strains used will protect against both lineages. In addition, for FMDV vaccine banks that usually hold a limited number of strains, it is necessary to include strains with a broad antigenic coverage. To assess the cross protective ability of an A/G-VII emergency vaccine (formulated at 43 (95% CI 8–230) PD50/dose as determined during homologous challenge), we performed a heterologous potency test according to the European Pharmacopoeia design using a field isolate from the A/IRN/05 lineage as the challenge virus. The estimated heterologous potency in this study was 2.0 (95% CI 0.4–6.0) PD50/dose, which is below the minimum potency recommended by the World Organisation for Animal Health (OIE). Furthermore, the cross-reactive antibody titres against the heterologous challenge virus were poor (≤log10 0.9), even in those cattle that had received the full dose of vaccine. The geometric mean r1-value was 0.2 (95% CI 0.03–0.8), similar to the potency ratio of 0.04 (95% CI 0.004–0.3). Vaccination decreased viraemia and virus excretion compared to the unvaccinated controls. Our results indicate that this A/G-VII vaccine does not provide sufficient protection against viruses belonging to the A/IRN/05 lineage and therefore the A/G-VII vaccine strain cannot replace the A/IRN/05 vaccine strain but could be considered an additional strain for use in vaccines and antigen banks.

SIV Evolutionary Dynamics in Cynomolgus Macaques during SIV-Mycobacterium tuberculosis Co-Infection

Co-infection with Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) is a worldwide public health concern, leading to worse clinical outcomes caused by both pathogens. We used a non-human primate model of simian immunodeficiency virus (SIV)-Mtb co-infection, in which latent Mtb infection was established prior to SIVmac251 infection. The evolutionary dynamics of SIV env was evaluated from samples in plasma, lymph nodes, and lungs (including granulomas) of SIV-Mtb co-infected and SIV only control animals. While the diversity of the challenge virus was low and overall viral diversity remained relatively low over 6–9 weeks, changes in viral diversity and divergence were observed, including evidence for tissue compartmentalization. Overall, viral diversity was highest in SIV-Mtb animals that did not develop clinical Mtb reactivation compared to animals with Mtb reactivation. Among lung granulomas, viral diversity was positively correlated with the frequency of CD4+ T cells and negatively correlated with the frequency of CD8+ T cells. SIV diversity was highest in the thoracic lymph nodes compared to other sites, suggesting that lymphatic drainage from the lungs in co-infected animals provides an advantageous environment for SIV replication. This is the first assessment of SIV diversity across tissue compartments during SIV-Mtb co-infection after established Mtb latency.

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2–neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P–immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

SARS-CoV-2 Virus like Particles produced by a single recombinant baculovirus generate potent neutralizing antibody that protects against variant challenge

The Covid-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for the rapid generation of efficient vaccines for emerging disease. Virus-like particles, VLPs, are an established vaccine technology that produces virus-like mimics, based on expression of the structural proteins of a target virus that can stimulate strong neutralizing antibody responses. SARS-CoV-2 is a coronavirus where the basis of VLP formation has been shown to be the co-expression of the spike, membrane and envelope structural proteins. Here we describe the generation of SARS-CoV-2 VLPs by the co expression of the salient structural proteins in insect cells using the established baculovirus expression system. VLPs were heterologous ~100nm diameter enveloped particles with a distinct fringe that reacted strongly with SARS-CoV-2 convalescent sera. In a Syrian hamster challenge model, a non adjuvanted VLPs induced neutralizing antibodies to the VLP-associated Wuhan S protein, reduced virus shedding following a virulent challenge with SARS-CoV-2 (B.1.1.7 variant) and protected against disease associated weight loss. Immunized animals showed reduced lung pathology and lower challenge virus replication than the non-immunized controls. Our data, using an established and scalable technology, suggest SARS-CoV-2 VLPs offer an efficient vaccine that mitigates against virus load and prevents severe disease.

Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

In vivo Serial Passaging of Human–Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

10. Quadrivalent M2SR (M2-deficient Single Replication) Live Influenza Vaccine Provides Better Protection Than Inactivated Vaccine Against Drifted Influenza B Virus Challenge in Ferrets

Background Quadrivalent inactivated influenza vaccines (QIV) induce neutralizing antibodies (Abs) against the viral hemagglutinin (HA). Despite annual update of HA vaccine antigens to match circulating strains, current vaccines provide ~60% vaccine effectiveness (VE). QIV VE can be as low as 10% when circulating strains do not match vaccine HA. The live M2SR (M2-deficient single replication) influenza vaccine candidate has previously shown broad humoral, mucosal and cellular immune responses and protection against multiple influenza A subtypes. Here we show similar properties with the Quadrivalent M2SR (Quad M2SR) against drifted influenza B challenge in comparison to QIV. Methods Ferrets pre-infected with influenza H1N1 and B/Yamagata viruses, were immunized intranasally (IN) with PBS (Mock) or Quad M2SR, or intramuscularly with Fluzone QIV. Serum collected post-vaccination was evaluated for Ab responses. Forty-two days after vaccination, ferrets were challenged IN with 106 pfu of B/Brisbane/60/2008 (Victoria lineage) influenza virus. Nasal washes were taken for 7 days post-challenge and evaluated for challenge virus by TCID50 assay. Nasal turbinates, trachea and lungs were also evaluated for virus. Results Quad M2SR and QIV elicited high serum Abs against the vaccine strain B/Colorado/06/2017 (Fig. 1A) and against the drifted influenza B challenge strain B/Brisbane/60/2008 (Fig. 1B) in ferrets with preexisting immunity. Like Mock, ferrets who received QIV displayed both weight loss (6.2%, Fig. 2A) and a rise in temperature (1.1oC, Fig. 2B) after challenge. In contrast, the Quad M2SR group did not exhibit any significant weight or temperature changes after challenge. Quad M2SR controlled the drifted challenge virus better than QIV as evidenced by significantly lower or absent post-challenge virus titer in nasal washes (Fig. 3A) and nasal turbinates (Fig. 3B). Figure 1. Serum Neutralization Titers Post-Vaccination Plaque reduction neutralization test (PRNT) antibody titers for Quad M2SR and QIV against matched Influenza B vaccine strain B/Colorado/06/2017 (Fig. 1A) and drifted strain B/Brisbane/60/2008 (Fig. 1B) on pre-study (Day -3), pre-vaccination (Day 28), and 3 weeks post vaccination (Day 51). The detection limit of the assay (horizontal dashed line) was 15 PRNT50. Figure 2. Post-challenge body weight and temperature changes Percent body weight changes (Fig. 2A) and average body temperatures changes (Fig. 2B) following challenge with drifted Influenza B strain B/Brisbane/60/2008 for ferrets vaccinated with Quad M2SR or QIV. Figure 3. Post-challenge virus titers in respiratory tract. Viral titers in nasal washes (Fig. 3A) and nasal turbinates (Fig. 3B) collected post-challenge with Influenza B strain B/Brisbane/60/2008 in ferrets vaccinated with Quad M2SR or QIV. No virus was detected in the trachea or lungs. The detection limit of the assay (horizontal dashed line) was 1.5 log10 TCID50/mL and 20 FFU respectively. Virus titer between groups was significant on day 3 of the nasal washes: one-way analysis of variance (ANOVA) with Multiple t tests to compare between groups, #p&lt;0.05,&gt;&lt;0.01,&gt;&lt;&gt; Conclusion Despite eliciting similar Ab titers, the Quad M2SR demonstrated superior protection compared to QIV in a drifted influenza B challenge model in ferrets. These results suggest that the intranasal M2SR platform may confer additional advantages over currently available vaccines. Quad M2SR is in late-stage development for testing in a first-in-human clinical study. Disclosures Lindsay Hill-Batorski, PhD, FluGen (Employee) Yasuko Hatta, DVM, PhD, FluGen (Employee) Michael Moser, PhD, FluGen (Employee) David Marshall, BS, FluGen (Employee) Pamuk Bilsel, PhD, FluGen (Employee)

Novel Recombinant Newcastle Disease Virus-Based In Ovo Vaccines Bypass Maternal Immunity to Provide Full Protection from Early Virulent Challenge

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple timepoints. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines provided protection from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that are able to penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

Immune Response in Mice Immunized with Chimeric H1 Antigens

Identification of a universal influenza vaccine candidate has remained a global challenge for both humans and animals. This study describes an approach that uses consensus sequence building to generate chimeric HAs (cHAs): two resultant H1 HA-based chimeras comprising of conserved sequences (within several areas spanning the head and stalk regions) of H1 and H5 or H9 HAs. These cHAs expressed in Drosophila cells (S2) were used to immunize mice. All immunized mice were protected from an infectious H1 virus challenge. Seroconverted mice sera to the H1 cHAs inhibited both the challenge virus and an H5 virus isolate by haemagglutination inhibition (HI) assay. These findings further emphasize that cHAs induce cross-reactive antibodies against conserved areas of both head and stalk regions of the seasonal influenza A (H1N1) pdm09 virus’ HA and holds potential for further development of a universal influenza vaccine.

Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence in blood and virulence in pigs infected with BeninΔDP148R

The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. Previously, the DP148R gene was deleted from the genome of genotype I virulent Benin 97/1 isolate. This virus, BeninΔDP148R, induced transient moderate clinical signs after immunization and high levels of protection against challenge. However, the BeninΔDP148R virus and genome persisted in blood over a prolonged period. In the current study deletion of either EP402R or EP153R genes individually or in combination from BeninΔDP148R genome was shown not to reduce virus replication in macrophages in vitro. However, deletion of EP402R dramatically reduced the period of infectious virus persistence in blood in immunized pigs from 28 to 14 days and virus genome from 59 to 14 days, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (BeninΔDP148RΔEP153RΔEP402R) further attenuated the virus and no viremia or clinical signs were observed post-immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from BeninΔDP148R did not result in further virus attenuation and did not reduce the period of virus persistence in blood. These results show that EP402R and EP153R have a synergistic role in reducing clinical signs and levels of virus in blood. Importance: African swine fever virus (ASFV) causes a disease of domestic pigs and wild boar which results in death of almost all infected animals. The disease has a high economic impact, and no vaccine is available. We investigated the role of two ASFV proteins, called EP402R and EP153R, in determining the levels and length of time virus persists in blood from infected pigs. EP402R causes ASFV particles and infected cells to bind to red blood cells. Deletion of the EP402R gene dramatically reduced virus persistence in blood but did not reduce the level of virus. Deletion of the EP153R alone did not reduce the period or level of virus persistence in blood. However, deleting both EP153R and EP402R resulted in undetectable levels of virus in blood and no clinical signs showing the proteins act synergistically. Importantly the infected pigs were protected following infection with the wildtype virus that kills pigs.