Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection.

The first exposure to influenza is presumed to shape the B-cell antibody repertoire, leading to preferential enhancement of the initially formed responses during subsequent exposure to viral variants. Here, we investigated whether this principle remains applicable when there are large genetic and antigenic differences between primary and secondary influenza virus antigens. Because humans usually have a complex history of influenza virus exposure, we conducted this investigation in influenza-naive cynomolgus macaques. Two groups of six macaques were immunized four times with influenza virus-like particles (VLPs) displaying either one (monovalent) or five (pentavalent) different hemagglutinin (HA) antigens derived from seasonal H1N1 (H1N1) strains. Four weeks after the final immunization, animals were challenged with pandemic H1N1 (H1N1pdm09). Although immunization resulted in robust virus-neutralizing responses to all VLP-based vaccine strains, there were no cross-neutralization responses to H1N1pdm09, and all animals became infected. No reductions in viral load in the nose or throat were detected in either vaccine group. After infection, strong virus-neutralizing responses to H1N1pdm09 were induced. However, there were no increases in virus-neutralizing titers against four of the five H1N1 vaccine strains; and only a mild increase was observed in virus-neutralizing titer against the influenza A/Texas/36/91 vaccine strain. After H1N1pdm09 infection, both vaccine groups showed higher virus-neutralizing titers against two H1N1 strains of intermediate antigenic distance between the H1N1 vaccine strains and H1N1pdm09, compared with the naive control group. Furthermore, both vaccine groups had higher HA-stem antibodies early after infection than the control group. In conclusion, immunization with VLPs displaying HA from antigenically distinct H1N1 variants increased the breadth of the immune response during subsequent H1N1pdm09 challenge, although this phenomenon was limited to intermediate antigenic variants.

Blocking NS3-NS4B interaction inhibits dengue virus in non-human primates.

Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.

A low dose of RBD and TLR7/8 agonist displayed on influenza virosome particles protects rhesus macaque against SARS-CoV-2 challenge.

Influenza virosomes serve as antigen delivery vehicles and pre-existing immunity toward influenza improves the immune responses toward antigens. Here, vaccine efficacy was evaluated in non-human primates with a COVID-19 virosome-based vaccine containing a low dose of RBD protein (15 µg) and the adjuvant 3M-052 (1 µg), displayed together on virosomes. Vaccinated animals (n = 6) received two intramuscular administrations at week 0 and 4 and challenged with SARS-CoV-2 at week 8, together with unvaccinated control animals (n = 4). The vaccine was safe and well tolerated and serum RBD IgG antibodies were induced in all animals and in the nasal washes and bronchoalveolar lavages in the three youngest animals. All control animals became strongly sgRNA positive in BAL, while all vaccinated animals were protected, although the oldest vaccinated animal (V1) was transiently weakly positive. The three youngest animals had also no detectable sgRNA in nasal wash and throat. Cross-strain serum neutralizing antibodies toward Wuhan-like, Alpha, Beta, and Delta viruses were observed in animals with the highest serum titers. Pro-inflammatory cytokines IL-8, CXCL-10 and IL-6 were increased in BALs of infected control animals but not in vaccinated animals. Virosomes-RBD/3M-052 prevented severe SARS-CoV-2, as shown by a lower total lung inflammatory pathology score than control animals.

Primary antibody response after influenza virus infection is first dominated by low-mutated HA-stem antibodies followed by higher-mutated HA-head antibodies.

Several studies have shown that the first encounter with influenza virus shapes the immune response to future infections or vaccinations. However, a detailed analysis of the primary antibody response is lacking as this is difficult to study in humans. It is therefore not known what the frequency and dynamics of the strain-specific hemagglutinin (HA) head- and stem-directed antibody responses are directly after primary influenza virus infection. Here, sera of twelve H1N1pdm2009 influenza virus-infected cynomolgus macaques were evaluated for HA-head and HA-stem domain antibody responses. We observed an early induction of HA-stem antibody responses, which was already decreased by day 56. In contrast, responses against the HA-head domain were low early after infection and increased at later timepoint. The HA-specific B cell repertoires in each animal showed diverse VH-gene usage with preferred VH-gene and JH-gene family usage for HA-head or HA-stem B cells but a highly diverse allelic variation within the VH-usage. HA-head B cells had shorter CDRH3s and higher VH-gene somatic hyper mutation levels relative to HA-stem B cells. In conclusion, our data suggest that HA-stem antibodies are the first to react to the infection while HA-head antibodies show a delayed response, but a greater propensity to enter the germinal center and undergo affinity maturation.

Safety and immunogenicity of four-segmented Rift Valley fever virus in the common marmoset.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild and domesticated ruminants, camelids, and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with contaminated tissues or blood. No human vaccine is available and commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating accidental exposure of humans to the veterinary vaccine and the application of hRVFV-4s to humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding, and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild-to-severe morbidity. In contrast, both vaccines were well tolerated with no evidence of dissemination and shedding while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

A Capsid Virus-Like Particle-Based SARS-CoV-2 Vaccine Induces High Levels of Antibodies and Protects Rhesus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. Here, we present non-human primate immunogenicity and protective efficacy data generated with the capsid virus-like particle (cVLP)-based vaccine ABNCoV2 that has previously demonstrated immunogenicity in mice. In rhesus macaques, a single vaccination with either 15 or 100 µg ABNCoV2 induced binding and neutralizing antibodies in a dose-dependent manner, at levels comparable to those measured in human convalescents. A second vaccine administration led to a >50-fold increase in neutralizing antibodies, with 2-log higher mean levels in the 100-µg ABNCoV2 group compared with convalescent samples. Upon SARS-CoV-2 challenge, a significant reduction in viral load was observed for both vaccine groups relative to the challenge control group, with no evidence of enhanced disease. Remarkably, neutralizing antibody titers against an original SARS-CoV-2 isolate and against variants of concern were comparable, indicating a potential for broad protection afforded by ABNCoV2, which is currently in clinical testing.

Brain Inflammation and Intracellular α-Synuclein Aggregates in Macaques after SARS-CoV-2 Infection.

SARS-CoV-2 causes acute respiratory disease, but many patients also experience neurological complications. Neuropathological changes with pronounced neuroinflammation have been described in individuals after lethal COVID-19, as well as in the CSF of hospitalized patients with neurological complications. To assess whether neuropathological changes can occur after a SARS-CoV-2 infection, leading to mild-to-moderate disease, we investigated the brains of four rhesus and four cynomolgus macaques after pulmonary disease and without overt clinical symptoms. Postmortem analysis demonstrated the infiltration of T-cells and activated microglia in the parenchyma of all infected animals, even in the absence of viral antigen or RNA. Moreover, intracellular α-synuclein aggregates were found in the brains of both macaque species. The heterogeneity of these manifestations in the brains indicates the virus' neuropathological potential and should be considered a warning for long-term health risks, following SARS-CoV-2 infection.

Poxvirus MVA Expressing SARS-CoV-2 S Protein Induces Robust Immunity and Protects Rhesus Macaques From SARS-CoV-2.

Novel safe, immunogenic, and effective vaccines are needed to control the COVID-19 pandemic, caused by SARS-CoV-2. Here, we describe the safety, robust immunogenicity, and potent efficacy elicited in rhesus macaques by a modified vaccinia virus Ankara (MVA) vector expressing a full-length SARS-CoV-2 spike (S) protein (MVA-S). MVA-S vaccination was well tolerated and induced S and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against SARS-CoV-2 and several variants of concern. S-specific IFNγ, but not IL-4, -producing cells were also elicited. After SARS-CoV-2 challenge, vaccinated animals showed a significant strong reduction of virus loads in bronchoalveolar lavages (BAL) and decreased levels in throat and nasal mucosa. Remarkably, MVA-S also protected macaques from fever and infection-induced cytokine storm. Computed tomography and histological examination of the lungs showed reduced lung pathology in MVA-S-vaccinated animals. These findings favor the use of MVA-S as a potential vaccine for SARS-CoV-2 in clinical trials.

Influenza A Virus Hemagglutinin Trimer, Head and Stem Proteins Identify and Quantify Different Hemagglutinin-Specific B Cell Subsets in Humans.

Antibody responses against the influenza A virus hemagglutinin (HA)-protein are studied intensively because they can protect against (re)infection. Previous studies have focused on antibodies targeting the head or stem domains, while other possible specificities are often not taken into account. To study such specificities, we developed a diverse set of HA-domain proteins based on an H1N1pdm2009-like influenza virus strain, including monomeric head and trimeric stem domain, as well as the full HA-trimer. These proteins were used to study the B cell and antibody responses in six healthy human donors. A large proportion of HA-trimer B cells bound exclusively to HA-trimer probe (54-77%), while only 8-18% and 9-23% were able to recognize the stem or head probe, respectively. Monoclonal antibodies (mAbs) were isolated and three of these mAbs, targeting the different domains, were characterized in-depth to confirm the binding profile observed in flow cytometry. The head-directed mAb, targeting an epitope distinct from known head-specific mAbs, showed relatively broad H1N1 neutralization and the stem-directed mAb was able to broadly neutralize diverse H1N1 viruses. Moreover, we identified a trimer-directed mAb that did not compete with known head or stem domain specific mAbs, suggesting that it targets an unknown epitope or conformation of influenza virus' HA. These observations indicate that the described method can characterize the diverse antibody response to HA and might be able to identify HA-specific B cells and antibodies with previously unknown specificities that could be relevant for vaccine design.

The Post-Acute Phase of SARS-CoV-2 Infection in Two Macaque Species Is Associated with Signs of Ongoing Virus Replication and Pathology in Pulmonary and Extrapulmonary Tissues.

The post-acute phase of SARS-CoV-2 infection was investigated in rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). During the acute phase of infection, SARS-CoV-2 was shed via the nose and throat, and viral RNA was occasionally detected in feces. This phase coincided with a transient change in systemic immune activation. Even after the alleged resolution of the infection, computed tomography (CT) and positron emission tomography (PET)-CT revealed pulmonary lesions and activated tracheobronchial lymph nodes in all animals. Post-mortem histological examination of the lung tissue revealed mostly marginal or resolving minimal lesions that were indicative of SARS-CoV-2 infection. Evidence for SARS-CoV-2-induced histopathology was also found in extrapulmonary tissue samples, such as conjunctiva, cervical, and mesenteric lymph nodes. However, 5-6 weeks after SARS-CoV-2 exposure, upon necropsy, viral RNA was still detectable in a wide range of tissue samples in 50% of the macaques and included amongst others the heart, the respiratory tract and surrounding lymph nodes, salivary gland, and conjunctiva. Subgenomic messenger RNA was detected in the lungs and tracheobronchial lymph nodes, indicative of ongoing virus replication during the post-acute phase. These results could be relevant for understanding the long-term consequences of COVID-19 in humans.

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.

Aerosolized Exposure to H5N1 Influenza Virus Causes Less Severe Disease Than Infection via Combined Intrabronchial, Oral, and Nasal Inoculation in Cynomolgus Macaques.

Infection with highly pathogenic avian H5N1 influenza virus in humans often leads to severe respiratory disease with high mortality. Experimental infection in non-human primates can provide additional insight into disease pathogenesis. However, such a model should recapitulate the disease symptoms observed in humans, such as pneumonia and inflammatory cytokine response. While previous studies in macaques have demonstrated the occurrence of typical lesions in the lungs early after infection and a high level of immune activation, progression to severe disease and lethality were rarely observed. Here, we evaluated a routinely used combined route of infection via intra-bronchial, oral, and intra-nasal virus inoculation with aerosolized H5N1 exposure, with or without the regular collection of bronchoalveolar lavages early after infection. Both combined route and aerosol exposure resulted in similar levels of virus replication in nose and throat and similar levels of immune activation, cytokine, and chemokine release in the blood. However, while animals exposed to H5N1 by combined-route inoculation developed severe disease with high lethality, aerosolized exposure resulted in less lesions, as measured by consecutive computed tomography and less fever and lethal disease. In conclusion, not virus levels or immune activation, but route of infection determines fatal outcome for highly pathogenic avian H5N1 influenza infection.

A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits.

BACKGROUND: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. METHOD: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. RESULTS: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. DISCUSSION: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate.

The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.

Aerosolized pH1N1 influenza infection induces less systemic and local immune activation in the lung than combined intrabronchial, nasal and oral exposure in cynomolgus macaques.

Non-human primates form an important animal model for the evaluation of immunogenicity and efficacy of novel 'universal' vaccine candidates against influenza virus. However, in most studies a combination of intra-tracheal or intra-bronchial, oral and nasal virus inoculation is used with a standard virus dose of between 1 and 10 million tissue culture infective doses, which differs from typical modes of virus exposure in humans. This paper studies the systemic and local inflammatory and immune effects of aerosolized versus combined-route exposure to pandemic H1N1 influenza virus. In agreement with a previous study, both combined-route and aerosol exposure resulted in similar levels of virus replication in nose, throat and lung lavages. However, the acute release of pro-inflammatory cytokines and chemokines, acute monocyte activation in peripheral blood as well as increased cytokine production and T-cell proliferation in the lungs were only observed after combined-route infection and not after aerosol exposure. Longitudinal evaluation by computed tomography demonstrated persistence of lung lesions after resolution of the infection and a tendency for more lesions in the lower lung lobes after combined-route exposure versus upper and middle lung lobes after aerosol exposure. Computed tomography scores were observed to correlate with fever. In conclusion, influenza virus infection by aerosol exposure is accompanied by less immune-activation and inflammation in comparison with direct virus installation, despite similar levels of virus replication and development of lesions in the lungs.

How the COVID-19 pandemic highlights the necessity of animal research.

Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternative methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and the various animal-free alternatives currently available do not come close to this complexity. In this Essay, we therefore argue that the use of animals is essential for the advancement of human and veterinary health.

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model.

The current pandemic coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), was recently identified in patients with an acute respiratory syndrome, coronavirus disease 2019 (COVID-19). To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or Middle East respiratory syndrome (MERS)-CoV and compared the pathology and virology with historical reports of SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in the absence of clinical signs and detected in type I and II pneumocytes in foci of diffuse alveolar damage and in ciliated epithelial cells of nasal, bronchial, and bronchiolar mucosae. In SARS-CoV infection, lung lesions were typically more severe, whereas they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 causes COVID-19-like disease in macaques and provides a new model to test preventive and therapeutic strategies.

Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (Macaca radiata).

BACKGROUND: Primate T-lymphotropic viruses type 1 (PTLV-1) are complex retroviruses infecting both human (HTLV-1) and simian (STLV-1) hosts. They share common epidemiological, clinical and molecular features. In addition to the canonical gag, pol, env retroviral genes, PTLV-1 purportedly encodes regulatory (i.e. Tax, Rex, and HBZ) and accessory proteins (i.e. P12/8, P13, P30). The latter have been found essential for viral persistence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a STLV-1 virus from a bonnet macaque (Macaca radiata-Mra18C9), a monkey from India. The complete sequence was obtained and phylogenetic analyses were performed. The Mra18C9 strain is highly divergent from the known PTLV-1 strains. Intriguingly, the Mra18C9 lacks the 3 accessory open reading frames. In order to determine if the absence of accessory proteins is specific to this particular strain, a comprehensive analysis of the complete PTLV-1 genomes available in Genbank was performed and found that the lack of one or many accessory ORF is common among PTLV-1. CONCLUSION: This study raises many questions regarding the actual nature, role and importance of accessory proteins in the PTLV-1 biology.

Structure and immunogenicity of a stabilized HIV-1 envelope trimer based on a group-M consensus sequence.

Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses.