Neuraminidase-Inhibiting Antibody Titers Correlate with Protection from Heterologous Influenza Virus Strains of the Same Neuraminidase Subtype.

Immune responses induced by currently licensed inactivated influenza vaccines are mainly directed against the hemagglutinin (HA) glycoprotein, the immunodominant antigen of influenza viruses. The resulting antigenic drift of HA requires frequent updating of the vaccine composition and annual revaccination. On the other hand, the levels of antibodies directed against the neuraminidase (NA) glycoprotein, the second major influenza virus antigen, vary greatly. To investigate the potential of the more conserved NA protein for the induction of subtype-specific protection, vesicular stomatitis virus-based replicons expressing a panel of N1 proteins from prototypic seasonal and pandemic H1N1 strains and human H5N1 and H7N9 isolates were generated. Immunization of mice and ferrets with the replicon carrying the matched N1 protein resulted in robust humoral and cellular immune responses and protected against challenge with the homologous influenza virus with an efficacy similar to that of the matched HA protein, illustrating the potential of the NA protein as a vaccine antigen. The extent of protection after immunization with mismatched N1 proteins correlated with the level of cross-reactive neuraminidase-inhibiting antibody titers. Passive serum transfer experiments in mice confirmed that these functional antibodies determine subtype-specific cross-protection. Our findings illustrate the potential of NA-specific immunity for achieving broader protection against antigenic drift variants or newly emerging viruses carrying the same NA but a different HA subtype.IMPORTANCE Despite the availability of vaccines, annual influenza virus epidemics cause 250,000 to 500,000 deaths worldwide. Currently licensed inactivated vaccines, which are standardized for the amount of the hemagglutinin (HA) antigen, primarily induce strain-specific antibodies, whereas the immune response to the neuraminidase (NA) antigen, which is also present on the viral surface, is usually low. Using NA-expressing single-cycle vesicular stomatitis virus replicons, we show that the NA antigen conferred protection of mice and ferrets against not only the matched influenza virus strains but also viruses carrying NA proteins from other strains of the same subtype. The extent of protection correlated with the level of cross-reactive NA-inhibiting antibodies. This highlights the potential of the NA antigen for the development of more broadly protective influenza vaccines. Such vaccines may also provide partial protection against newly emerging strains with the same NA but a different HA subtype.

Innate Responses to the Former COVID-19 Vaccine Candidate CVnCoV and Their Relation to Reactogenicity and Adaptive Immunogenicity.

Vaccines are highly effective at preventing severe coronavirus disease (COVID-19). With mRNA vaccines, further research is needed to understand the association between immunogenicity and reactogenicity, which is defined as the physical manifestation of an inflammatory response to a vaccination. This study analyzed the immune response and reactogenicity in humans, post immunization, to the former SARS-CoV-2 mRNA investigational vaccine CVnCoV (CV-NCOV-001 and CV-NCOV-002 clinical trials). Immunogenicity was investigated using whole-blood RNA sequencing, serum cytokine levels, and SARS-CoV-2-specific antibodies. The T cell responses in peripheral blood were assessed using intracellular cytokine staining (ICS) and high-dimensional profiling in conjunction with SARS-CoV-2 antigen-specificity testing via mass cytometry. Reactogenicity was graded after participants' first and second doses of CVnCoV using vaccine-related solicited adverse events (AEs). Finally, a Spearman correlation was performed between reactogenicity, humoral immunity, and serum cytokine levels to assess the relationship between reactogenicity and immunogenicity post CVnCoV vaccination. Our findings showed that the gene sets related to innate and inflammatory immune responses were upregulated one day post CVnCoV vaccination, while the gene sets related to adaptive immunity were upregulated predominantly one week after the second dose. The serum levels of IFNα, IFNγ, IP-10, CXCL11, IL-10, and MCP-1 increased transiently, peaking one day post vaccination. CD4+ T cells were induced in all vaccinated participants and low frequencies of CD8+ T cells were detected by ex vivo ICS. Using mass cytometry, SARS-CoV-2 spike-specific CD8+ T cells were induced and were characterized as having an activated effector memory phenotype. Overall, the results demonstrated a positive correlation between vaccine-induced systemic cytokines, reactogenicity, and adaptive immunity, highlighting the importance of the balance between the induction of innate immunity to achieve vaccine efficacy and ensuring low reactogenicity.

A protective measles virus-derived vaccine inducing long-lasting immune responses against influenza A virus H7N9.

A novel Influenza A virus (subtype H7N9) emerged in spring 2013 and caused considerable mortality in zoonotically infected patients. To be prepared for potential pandemics, broadly effective and safe vaccines are crucial. Recombinant measles virus (MeV) encoding antigens of foreign pathogens constitutes a promising vector platform to generate novel vaccines. To characterize the efficacy of H7N9 antigens in a prototypic vaccine platform technology, we generated MeVs encoding either neuraminidase (N9) or hemagglutinin (H7). Moraten vaccine strain-derived vaccine candidates were rescued; they replicated with efficiency comparable to that of the measles vaccine, robustly expressed H7 and N9, and were genetically stable over 10 passages. Immunization of MeV-susceptible mice triggered the production of antibodies against H7 and N9, including hemagglutination-inhibiting and neutralizing antibodies induced by MVvac2-H7(P) and neuraminidase-inhibiting antibodies by MVvac2-N9(P). Vaccinated mice also developed long-lasting H7- and N9-specific T cells. Both MVvac2-H7(P) and MVvac2-N9(P)-vaccinated mice were protected from lethal H7N9 challenge.

Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia.

Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine formulated with lipid nanoparticles in human volunteers: A phase 1 trial.

INTRODUCTION: In a first-in-human study immune responses to rabies virus glycoprotein (RABV-G)-mRNA vaccine were dependent on the route of administration, necessitating specialized devices. Following successful preclinical studies with mRNA encapsulated in lipid nanoparticles (LNP), we tested an mRNA-LNP formulation (CV7202). METHODS: In this phase 1, multi-center, controlled study in Belgium and Germany we enrolled 55 healthy 18-40-year-olds to receive intramuscular injections of 5 µg (n = 10), 1 µg (n = 16), or 2 µg (n = 16) CV7202 on Day 1; subsets (n = 8) of 1 µg and 2 µg groups received second doses on Day 29. Controls (n = 10) received rabies vaccine, Rabipur, on Days 1, 8 and 29. Safety and reactogenicity were assessed up to 28 days post-vaccination using diary cards; immunogenicity was measured as RABV-G-specific neutralizing titers (VNT) by RFFIT and IgG by ELISA. RESULTS: As initially tested doses of 5 µg CV7202 elicited unacceptably high reactogenicity we subsequently tested 1 and 2 µg doses which were better tolerated. No vaccine-related serious adverse events or withdrawals occurred. Low, dose-dependent VNT responses were detectable from Day 15 and by Day 29%, 31% and 22% of 1, 2 and 5 µg groups, respectively, had VNTs ≥ 0·5 IU/mL, considered an adequate response by the WHO. After two 1 or 2 µg doses all recipients had titers ≥ 0.5 IU/mL by Day 43. Day 57 GMTs were not significantly lower than those with Rabipur, which elicited adequate responses in all vaccinees after two doses. CV7202-elicited VNT were significantly correlated with RABV-G-specific IgG antibodies (r2 = 0.8319, p < 0.0001). CONCLUSIONS: Two 1 µg or 2 µg doses of CV7202 were well tolerated and elicited rabies neutralizing antibody responses that met WHO criteria in all recipients, but 5 µg had unacceptable reactogenicity for a prophylactic vaccine. ClinicalTrials.gov Identifier: NCT03713086.

Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2 : A phase 1 randomized clinical trial.

BACKGROUND: We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV­2 spike (S) protein encapsulated in lipid nanoparticles (LNP). METHODS: This is an interim analysis of a dosage escalation phase 1 study in healthy 18-60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV­2 S­protein and receptor binding domain (RBD), and SARS-CoV­2 neutralizing titers (MN50). RESULTS: In 245 volunteers who received 2 CVnCoV vaccinations (2 µg, n = 47, 4 µg, n = 48, 6 µg, n = 46, 8 µg, n = 44, 12 µg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S­protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S­protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 µg group. Responses to 12 µg were comparable to those observed in convalescent sera from known COVID-19 patients. CONCLUSION: In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 µg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.

Adeno-associated virus-vectored influenza vaccine elicits neutralizing and Fcγ receptor-activating antibodies.

The current seasonal inactivated influenza vaccine protects only against a narrow range of virus strains as it triggers a dominant antibody response toward the hypervariable hemagglutinin (HA) head region. The discovery of rare broadly protective antibodies against conserved regions in influenza virus proteins has propelled research on distinct antigens and delivery methods to efficiently induce broad immunity toward drifted or shifted virus strains. Here, we report that adeno-associated virus (AAV) vectors expressing influenza virus HA or chimeric HA protected mice against homologous and heterologous virus challenges. Unexpectedly, immunization even with wild-type HA induced antibodies recognizing the HA-stalk and activating FcγR-dependent responses indicating that AAV-vectored expression balances HA head- and HA stalk-specific humoral responses. Immunization with AAV-HA partially protected also ferrets against a harsh virus challenge. Results from this study provide a rationale for further clinical development of AAV vectors as influenza vaccine platform, which could benefit from their approved use in human gene therapy.

Incomplete genetic reconstitution of B cell pools contributes to prolonged immunosuppression after measles.

Measles is a disease caused by the highly infectious measles virus (MeV) that results in both viremia and lymphopenia. Lymphocyte counts recover shortly after the disappearance of measles-associated rash, but immunosuppression can persist for months to years after infection, resulting in increased incidence of secondary infections. Animal models and in vitro studies have proposed various immunological factors underlying this prolonged immune impairment, but the precise mechanisms operating in humans are unknown. Using B cell receptor (BCR) sequencing of human peripheral blood lymphocytes before and after MeV infection, we identified two immunological consequences from measles underlying immunosuppression: (i) incomplete reconstitution of the naïve B cell pool leading to immunological immaturity and (ii) compromised immune memory to previously encountered pathogens due to depletion of previously expanded B memory clones. Using a surrogate model of measles in ferrets, we investigated the clinical consequences of morbillivirus infection and demonstrated a depletion of vaccine-acquired immunity to influenza virus, leading to a compromised immune recall response and increased disease severity after secondary influenza virus challenge. Our results show that MeV infection causes changes in naïve and memory B lymphocyte diversity that persist after the resolution of clinical disease and thus contribute to compromised immunity to previous infections or vaccinations. This work highlights the importance of MeV vaccination not only for the control of measles but also for the maintenance of herd immunity to other pathogens, which can be compromised after MeV infection.

Did you drink alcohol during pregnancy? Inaccuracy and discontinuity of women's self-reports: On the way to establish meconium ethyl glucuronide (EtG) as a biomarker for alcohol consumption during pregnancy.

Consuming alcohol during pregnancy is one of the most verified prenatal risk factors for impaired child development. Information about the amount of alcohol consumed prenatally is needed to anticipate negative effects and to offer timely support. Women's self-reports are not reliable, often influenced by social stigmas and retrospective recall bias, causing biomarkers of intrauterine ethanol exposure to become more and more relevant. The present study compares both women's gestational and retrospective self-reports of prenatal alcohol consumption with levels of ethyl glucuronide (EtG) in meconium. Women (n = 180) gave self-reports of prenatal alcohol consumption both during their 3rd trimester (gestational self-report) and when their children were 6-8 years old (retrospective self-report). Child meconium was collected after birth and analyzed for EtG. No individual feedback of children's EtG level was given to the women. All analyses were run separately for two cut-offs: 10 ng/g (limit of detection) and 120 ng/g (established by Goecke et al., 2014). Mothers of children with EtG values above 10 ng/g (n = 42) tended to report prenatal alcohol consumption more frequently. There was no trend or significance for the EtG cut-off of 120 ng/g (n = 26) or for retrospective self-report. When focusing on women who retrospectively reported alcohol consumption during pregnancy, a claim to five or more consumed glasses per month made an EtG over the 10 ng/g and the 120 ng/g cut-off more probable. Women whose children were over the 10 ng/g EtG cut-off were the most inconsistent in their self-report behavior, whereas the consistency in the above 120 ng/g EtG group was higher than in any other group. The next step to establish EtG as a biomarker for intrauterine alcohol exposure is to correlate EtG values in meconium with child developmental impairments.

Novel Furin Inhibitors with Potent Anti-infectious Activity.

New peptidomimetic furin inhibitors with unnatural amino acid residues in the P3 position were synthesized. The most potent compound 4-guanidinomethyl-phenylacteyl-Arg-Tle-Arg-4-amidinobenzylamide (MI-1148) inhibits furin with a Ki value of 5.5 pM. The derivatives also strongly inhibit PC1/3, whereas PC2 is less affected. Selected inhibitors were tested in cell culture for antibacterial and antiviral activity against infectious agents known to be dependent on furin activity. A significant protective effect against anthrax and diphtheria toxin was observed in the presence of the furin inhibitors. Furthermore, the spread of the highly pathogenic H5N1 and H7N1 avian influenza viruses and propagation of canine distemper virus was strongly inhibited. Inhibitor MI-1148 was crystallized in complex with human furin. Its N-terminal guanidinomethyl group in the para position of the P5 phenyl ring occupies the same position as that found previously for a structurally related inhibitor containing this substitution in the meta position, thereby maintaining all of the important P5 interactions. Our results confirm that the inhibition of furin is a promising strategy for a short-term treatment of acute infectious diseases.