Optimizing a linear 'Doggybone' DNA vaccine for influenza virus through the incorporation of DNA targeting sequences and neuraminidase antigen.

Influenza virus represents a challenge for traditional vaccine approaches due to its seasonal changes and potential for zoonotic transmission. Nucleic acid vaccines can overcome some of these challenges, especially through the inclusion of multiple antigens to increase the breadth of response. RNA vaccines were an important part of the response to the COVID-19 pandemic, but for future outbreaks DNA vaccines may have some advantages in terms of stability and manufacturing cost that warrant continuing investigation to fully realize their potential. Here, we investigate influenza virus vaccines made using a closed linear DNA platform, Doggybone™ DNA (dbDNA), produced by a rapid and scalable cell-free method. Influenza vaccines have mostly focussed on Haemagglutinin (HA), but the inclusion of Neuraminidase (NA) may provide additional protection. Here, we explored the potential of including NA in a dbDNA vaccine, looking at DNA optimization, mechanism and breadth of protection. We showed that DNA targeting sequences (DTS) improved immune responses against HA but not NA. We explored whether NA vaccine-induced protection against influenza virus infection was cell-mediated, but depletion of CD8 and NK cells made no impact, suggesting it was antibody-mediated. This is reflected in the restriction of protection to homologous strains of influenza virus. Importantly, we saw that including both HA and NA in a single combined vaccine did not dampen the immune response to either one. Overall, we show that linear dbDNA can induce an immune response against NA, which may offer increased protection in instances of HA mismatch where NA remains more conserved.

Validation of a double-color ELISpot assay of IFN-γ and IL-4 production in human peripheral blood mononuclear cells.

The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest.

Adequate immune responses to vaccines after chemotherapy for leukaemia diagnosed in childhood.

AIM: The survival rate after treatment for childhood leukaemia has greatly improved, but could result in protracted immune deficiency. This study examined the immune status of children after chemotherapy and evaluated their responses to immunisation. METHODS: Subjects who had completed their treatment for acute lymphoblastic leukaemia at The Children's Hospital Reykjavík, Iceland, during 2011-2020 had blood drawn and were then immunised for influenza in October 2021. Blood was drawn again 4 weeks later and their humoral and cellular responses were measured with a haemagglutination inhibition assay and lymphocyte stimulation test. Antibodies to other immunisations were also evaluated. RESULTS: We studied 18 patients (10 male) who had completed their treatment at 3.7-20.3 years of age (mean 9.1), 11-84 months (mean 36.9) before enrolment. Conventional immunological evaluation did not reveal notable abnormalities. The responses to several childhood vaccinations, including the pneumococcal conjugate vaccination, were adequate in most patients. Humoral responses to the influenza vaccine confirmed adequate reactions in all but one patient. Considerable variations were observed in the lymphocyte stimulations tests. CONCLUSION: Most patients reacted adequately to immunisation, especially against annual influenza and Streptococcus pneumoniae, reiterating the usefulness of vaccinations. The most appropriate timing for vaccination after treatment still needs to be determined.

Obesity in adolescents does not influence early immune responses to influenza vaccination.

BACKGROUND: Obesity has been linked to reduced vaccine responses against tetanus, hepatitis B and influenza. Data on the influence of paediatric obesity on influenza vaccine response is still lacking and this study aims to fill the gap. METHODS: A total of 30 children with obesity and 30 children with normal weight, aged 12-18 years, were recruited. Participants were vaccinated with a tetravalent influenza vaccine. Blood was collected prior to the vaccination and again four weeks later. The humoral response was assessed with haemagglutinin inhibition assay. The cellular response was assessed with T-cell stimulation assays measuring TNF-α, IFN-γ, IL-2 and IL-13. RESULTS: Of the 29/30 from the study group and 30/30 from the control group finished both visits. Seroconversion occurred for > 90% of participants in both groups for the A/H1N1, A/H3N2 and B/Victoria strains, but the B/Yamagata strain had lower seroconversion rates (93% in the study group and 80% in the control group). 97-100% of participants from both groups had adequate serological responses following vaccination. Cellular responses were similar between the two groups post-vaccination. CONCLUSIONS: Early humoral and cellular immune responses to influenza vaccinations are similar among adolescents with obesity and normal weight.

Efficacy of mucosal vaccination using a protozoan parasite as a vehicle for antigen delivery: IgG and neutralizing response after rectal administration of LeCoVax-2, a candidate vaccine against COVID-19.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation.

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples.

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease. The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects. In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities.

Influenza Anti-Stalk Antibodies: Development of a New Method for the Evaluation of the Immune Responses to Universal Vaccine.

Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches.