Corrigendum: Targeting neoantigens to APC-surface molecules improves the immunogenicity and anti-tumor efficacy of a DNA cancer vaccine.

[This corrects the article DOI: 10.3389/fimmu.2023.1234912.].

Targeting neoantigens to APC-surface molecules improves the immunogenicity and anti-tumor efficacy of a DNA cancer vaccine.

Introduction: Tumor-specific mutations generate neoepitopes unique to the cancer that can be recognized by the immune system, making them appealing targets for therapeutic cancer vaccines. Since the vast majority of tumor mutations are patient-specific, it is crucial for cancer vaccine designs to be compatible with individualized treatment strategies. Plasmid DNA vaccines have substantiated the immunogenicity and tumor eradication capacity of cancer neoepitopes in preclinical models. Moreover, early clinical trials evaluating personalized neoepitope vaccines have indicated favorable safety profiles and demonstrated their ability to elicit specific immune responses toward the vaccine neoepitopes. Methods: By fusing in silico predicted neoepitopes to molecules with affinity for receptors on the surface of APCs, such as chemokine (C-C motif) ligand 19 (CCL19), we designed an APC-targeting cancer vaccine and evaluated their ability to induce T-cell responses and anti-tumor efficacy in the BALB/c syngeneic preclinical tumor model. Results: In this study, we demonstrate how the addition of an antigen-presenting cell (APC) binding molecule to DNA-encoded cancer neoepitopes improves neoepitope-specific T-cell responses and the anti-tumor efficacy of plasmid DNA vaccines. Dose-response evaluation and longitudinal analysis of neoepitope-specific T-cell responses indicate that combining APC-binding molecules with the delivery of personalized tumor antigens holds the potential to improve the clinical efficacy of therapeutic DNA cancer vaccines. Discussion: Our findings indicate the potential of the APC-targeting strategy to enhance personalized DNA cancer vaccines while acknowledging the need for further research to investigate its molecular mechanism of action and to translate the preclinical results into effective treatments for cancer patients.

SARS-CoV-2: Immunity, Challenges with Current Vaccines, and a Novel Perspective on Mucosal Vaccines.

The global rollout of COVID-19 vaccines has played a critical role in reducing pandemic spread, disease severity, hospitalizations, and deaths. However, the first-generation vaccines failed to block severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission, partially due to the limited induction of mucosal immunity, leading to the continuous emergence of variants of concern (VOC) and breakthrough infections. To meet the challenges from VOC, limited durability, and lack of mucosal immune response of first-generation vaccines, novel approaches are being investigated. Herein, we have discussed the current knowledge pertaining to natural and vaccine-induced immunity, and the role of the mucosal immune response in controlling SARS-CoV2 infection. We have also presented the current status of the novel approaches aimed at eliciting both mucosal and systemic immunity. Finally, we have presented a novel adjuvant-free approach to elicit effective mucosal immunity against SARS-CoV-2, which lacks the safety concerns associated with live-attenuated vaccine platforms.

Neuraminidase delivered as an APC-targeted DNA vaccine induces protective antibodies against influenza.

Conventional influenza vaccines focus on hemagglutinin (HA). However, antibody responses to neuraminidase (NA) have been established as an independent correlate of protection. Here, we introduced the ectodomain of NA into DNA vaccines that, as translated dimeric vaccine proteins, target antigen-presenting cells (APCs). The targeting was mediated by an single-chain variable fragment specific for major histocompatibility complex (MHC) class II, which is genetically linked to NA via a dimerization motif. A single immunization of BALB/c mice elicited strong and long-lasting NA-specific antibodies that inhibited NA enzymatic activity and reduced viral replication. Vaccine-induced NA immunity completely protected against a homologous influenza virus and out-competed NA immunity induced by a conventional inactivated virus vaccine. The protection was mainly mediated by antibodies, although NA-specific T cells also contributed. APC-targeting and antigen bivalency were crucial for vaccine efficacy. The APC-targeted vaccine was potent at low doses of DNA, indicating a dose-sparing effect. Similar results were obtained with NA vaccines that targeted different surface molecules on dendritic cells. Interestingly, the protective efficacy of NA as antigen compared favorably with HA and therefore ought to receive more attention in influenza vaccine research.

APC-Targeted DNA Vaccination Against Reticulocyte-Binding Protein Homolog 5 Induces Plasmodium falciparum-Specific Neutralizing Antibodies and T Cell Responses.

Targeted delivery of antigen to antigen presenting cells (APCs) is an efficient way to induce robust antigen-specific immune responses. Here, we present a novel DNA vaccine that targets the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5), a leading blood-stage antigen of the human malaria pathogen, to APCs. The vaccine is designed as bivalent homodimers where each chain is composed of an amino-terminal single chain fragment variable (scFv) targeting unit specific for major histocompatibility complex class II (MHCII) expressed on APCs, and a carboxyl-terminal antigenic unit genetically linked by the dimerization unit. This vaccine format, named "Vaccibody", has previously been successfully applied for antigens from other infectious diseases including influenza and HIV, as well as for tumor antigens. Recently, the crystal structure and key functional antibody epitopes for the truncated version of PfRH5 (PfRH5ΔNL) were characterized, suggesting PfRH5ΔNL to be a promising candidate for next-generation PfRH5 vaccine design. In this study, we explored the APC-targeting strategy for a PfRH5ΔNL-containing DNA vaccine. BALB/c mice immunized with the targeted vaccine induced higher PfRH5-specific IgG1 antibody responses than those vaccinated with a non-targeted vaccine or antigen alone. The APC-targeted vaccine also efficiently induced rapid IFN-γ and IL-4 T cell responses. Furthermore, the vaccine-induced PfRH5-specific IgG showed inhibition of growth of the P. falciparum 3D7 clone parasite in vitro. Finally, sera obtained after vaccination with this targeted vaccine competed for the same epitopes as PfRH5-specific mAbs from vaccinated humans. Robust humoral responses were also induced by a similar P. vivax Duffy-binding protein (PvDBP)-containing targeted DNA vaccine. Our data highlight a novel targeted vaccine platform for the development of vaccines against blood-stage malaria.

Pandemic Preparedness Against Influenza: DNA Vaccine for Rapid Relief.

The 2009 "swine flu" pandemic outbreak demonstrated the limiting capacity for egg-based vaccines with respect to global vaccine supply within a timely fashion. New vaccine platforms that efficiently can quench pandemic influenza emergences are urgently needed. Since 2009, there has been a profound development of new vaccine platform technologies with respect to prophylactic use in the population, including DNA vaccines. These vaccines are particularly well suited for global pandemic responses as the DNA format is temperature stable and the production process is cheap and rapid. Here, we show that by targeting influenza antigens directly to antigen presenting cells (APC), DNA vaccine efficacy equals that of conventional technologies. A single dose of naked DNA encoding hemagglutinin (HA) from influenza/A/California/2009 (H1N1), linked to a targeting moiety directing the vaccine to major histocompatibility complex class II (MHCII) molecules, raised similar humoral immune responses as the adjuvanted split virion vaccine Pandemrix, widely administered in the 2009 pandemic. Both vaccine formats rapidly induced serum antibodies that could protect mice already 8 days after a single immunization, in contrast to the slower kinetics of a seasonal trivalent inactivated influenza vaccine (TIV). Importantly, the DNA vaccine also elicited cytotoxic T-cell responses that reduced morbidity after vaccination, in contrast to very limited T-cell responses seen after immunization with Pandemrix and TIV. These data demonstrate that DNA vaccines has the potential as a single dose platform vaccine, with rapid protective effects without the need for adjuvant, and confirms the relevance of naked DNA vaccines as candidates for pandemic preparedness.

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses.

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus. Although both strategies are well-established, they have not previously been compared using the same system. We found that both antibody-antigen fusion modes led to presentation of the T-cell epitope. The strength of the T-cell responses varied, however, with the most efficient Troybody inducing CD4 T-cell proliferation and cytokine secretion at 10-100-fold lower concentration than the antibodies carrying antigen fused to the C-terminus, both in vitro and after intravenous injection in mice. Furthermore, we exchanged this loop with an MHCI-restricted T-cell epitope, and the resulting antibody enabled efficient cross-presentation to CD8 T cells in vivo. Targeting of antigen to APCs by use of such antibody-antigen fusions is thus an attractive vaccination strategy for increased activation of both CD4 and CD8 peptide-specific T cells.

Pustulan Activates Chicken Bone Marrow-Derived Dendritic Cells In Vitro and Promotes Ex Vivo CD4+ T Cell Recall Response to Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) is a highly contagious avian coronavirus. IBV causes substantial worldwide economic losses in the poultry industry. Vaccination with live-attenuated viral vaccines, therefore, are of critical importance. Live-attenuated viral vaccines, however, exhibit the potential for reversion to virulence and recombination with virulent field strains. Therefore, alternatives such as subunit vaccines are needed together with the identification of suitable adjuvants, as subunit vaccines are less immunogenic than live-attenuated vaccines. Several glycan-based adjuvants directly targeting mammalian C-type lectin receptors were assessed in vitro using chicken bone marrow-derived dendritic cells (BM-DCs). The ß-1-6-glucan, pustulan, induced an up-regulation of MHC class II (MHCII) cell surface expression, potentiated a strong proinflammatory cytokine response, and increased endocytosis in a cation-dependent manner. Ex vivo co-culture of peripheral blood monocytes from IBV-immunised chickens, and BM-DCs pulsed with pustulan-adjuvanted recombinant IBV N protein (rN), induced a strong recall response. Pustulan-adjuvanted rN induced a significantly higher CD4+ blast percentage compared to either rN, pustulan or media. However, the CD8+ and TCRγδ+ blast percentage were significantly lower with pustulan-adjuvanted rN compared to pustulan or media. Thus, pustulan enhanced the efficacy of MHCII antigen presentation, but apparently not the cross-presentation on MHCI. In conclusion, we found an immunopotentiating effect of pustulan in vitro using chicken BM-DCs. Thus, future in vivo studies might show pustulan as a promising glycan-based adjuvant for use in the poultry industry to contain the spread of coronaviridiae as well as of other avian viral pathogens.

A DNA Vaccine That Encodes an Antigen-Presenting Cell-Specific Heterodimeric Protein Protects against Cancer and Influenza.

Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen-presenting cells (APCs). In this study, we have developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE or the Barnase/Barstar motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity, and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins.

Targeting of HA to chemokine receptors induces strong and cross-reactive T cell responses after DNA vaccination in pigs.

Efficient influenza vaccination of pigs can reduce disease burdens for the swine industry, but also represents an important measure for reducing the risk from novel viral reassortments that pose pandemic threats to the human population. Here, we have vaccinated pigs with a DNA vaccine encoding influenza virus hemagglutinin (HA) linked to the chemokine MIP1α that bind chemokine receptors 1, 3, and 5 expressed on antigen presenting cells (APC). Such MIP1α targeting of HA to APC enhanced induction of HA reactive antibodies, particularly IgG2. In addition, the MIP1α- HA vaccine induced strong T cell responses that could cross-react with different influenza subtypes. Thus, the strategy of targeting HA to chemokine receptors could be important for inducing broad protection against antigenically diverse influenza strains in pigs.

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza.

Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza.IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells.

Targeting of nucleoprotein to chemokine receptors by DNA vaccination results in increased CD8(+)-mediated cross protection against influenza.

Vaccination is at present the most efficient way of preventing influenza infections. Currently used inactivated influenza vaccines can induce virus-neutralizing antibodies that are protective against a particular influenza strain, but hamper the induction of cross-protective T-cell responses to later infections. Thus, influenza vaccines need to be updated annually in order to confer protection against circulating influenza strains. This study aims at developing an efficient vaccine that can induce broader protection against influenza. For this purpose, we have used the highly conserved nucleoprotein (NP) from an influenza A virus subtype H7N7 strain, and inserted it into a vaccine format that targets an antigen directly to relevant antigen presenting cells (APCs). The vaccine format consists of bivalent antigenic and targeting units, linked via an Ig-based dimerization unit. In this study, NP was linked to MIP-1α, a chemokine that targets the linked antigen to chemokine receptors 1, 3 and 5 expressed on various APCs. The vaccine protein was indirectly delivered by DNA. Mice were vaccinated intradermally with plasmids, in combination with electroporation to enhance cellular uptake of DNA. We found that a single DNA vaccination was sufficient for induction of both antibody and T cell responses in BALB/c mice. Targeting of nucleoprotein to chemokine receptors enhanced T cell responses but not antibody responses. Moreover, a single dose of MIP1α-NP conferred protection in BALB/c mice against a lethal challenge with an H1N1 influenza virus. The observed cross-protection was mediated by CD8(+) T cells.

APC targeting enhances immunogenicity of a novel multistage Fc-fusion tuberculosis vaccine in mice.

Numerous studies have demonstrated that targeting immunogens to FcγR on antigen-presenting cells (APCs) can selectively uptake and increase cellular immunity in vitro and in vivo. Therefore, the present study was conducted to evaluate immunogenicity of a novel multistage tuberculosis vaccine, a combination of an early and a dormant immunogenic protein, ESAT6 and HspX, fused to Fcγ2a fragment of mouse IgG2a to target all forms of tuberculosis. Codon-optimized genes consisting of ESAT6, a linker, and HspX fused either to mouse Fcγ2a (ESAT6:HspX:mFcγ2a) or 6× His-tag (ESAT6:HspX:His) were synthesized. The resulting proteins were then produced in Pichia pastoris. The fusion proteins were separately emulsified in dimethyldioctadecylammonium bromide(DDA)-trehalose-6,6-dibehenate(TDB) adjuvant, and their immunogenicity with and without bacille Calmette-Guérin (BCG) was assessed in C57BL/6 mice. Th1, Th2, Th17, and T-reg cytokine patterns were evaluated using the ELISA method. Both multistage vaccines induced very strong IL-12 and IFN-γ secretion from splenic cells; the Fc-tagged subunit vaccine induced a more effective Th1 immune response (IFN-γ, 910 pg/mL, and IL-12, 854 pg/mL) with a very low increase in IL-17 (∼0.1 pg/mL) and IL-4 (37 pg/mL) and a mild increase in TGF-ß (543 pg/mL) compared to the BCG or ESAT6:HspX:His primed and boosted groups. The production of IFN-γ to ESAT6:HspX:Fcγ2a was very consistent and showed an increasing trend for IL-12 compared to the BCG or ESAT6:HspX:His primed and boosted groups. Fcγ2a used as a delivery vehicle supported the idea of selective uptake, inducing cross-presentation and forming a proper anti-tuberculosis response in context of Th1/Th2 and Th17/T-reg balances, which is important for protection and prevention of damage.

Polarizing T and B Cell Responses by APC-Targeted Subunit Vaccines.

Current influenza vaccines mostly aim at the induction of specific neutralizing antibodies. While antibodies are important for protection against a particular virus strain, T cells can recognize epitopes that will offer broader protection against influenza. We have previously developed a DNA vaccine format by which protein antigens can be targeted specifically to receptors on antigen presenting cells (APCs). The DNA-encoded vaccine proteins are homodimers, each chain consisting of a targeting unit, a dimerization unit, and an antigen. The strategy of targeting antigen to APCs greatly enhances immune responses as compared to non-targeted controls. Furthermore, targeting of antigen to different receptors on APCs can polarize the immune response to different arms of immunity. Here, we discuss how targeting of hemagglutinin to MHC class II molecules increases Th2 and IgG1 antibody responses, whereas targeting to chemokine receptors XCR1 or CCR1/3/5 increases Th1 and IgG2a responses, in addition to CD8(+) T cell responses. We also discuss these results in relation to work published by others on APC-targeting. Differential targeting of APC surface molecules may allow the induction of tailor-made phenotypes of adaptive immune responses that are optimal for protection against various infectious agents, including influenza virus.

Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.