A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Nonclinical safety evaluation of two vaccine candidates for herpes simplex virus type 2 to support combined administration in humans.

Herpes simplex virus type 2 (HSV-2) is the most common cause of genital disease worldwide. The development of an effective HSV-2 vaccine would significantly impact global health based on the psychological distress caused by genital herpes for some individuals, the risk transmitting the infection from mother to infant, and the elevated risk of acquiring HIV-1. Five nonclinical safety studies were conducted with the replication defective HSV529 vaccine, alone or adjuvanted with GLA-SE, and the G103 subunit vaccine containing GLA-SE. A biodistribution study was conducted in guinea pigs to evaluate distribution, persistence, and shedding of HSV529. A preliminary immunogenicity study was conducted in rabbits to demonstrate HSV529-specific humoral response and its enhancement by GLA-SE. Three repeated-dose toxicity studies, one in guinea pigs and two in rabbits, were conducted to assess systemic toxicity and local tolerance of HSV529, alone or adjuvanted with GLA-SE, or G103 containing GLA-SE. Data from these studies show that both vaccines are safe and well tolerated and support the ongoing HSV-2 clinical trial in which the two vaccine candidates will be given either sequentially or concomitantly to explore their potential synergistic and incremental effects.

HIV-1 Conserved Mosaics Delivered by Regimens with Integration-Deficient DC-Targeting Lentiviral Vector Induce Robust T Cells.

To be effective against HIV type 1 (HIV-1), vaccine-induced T cells must selectively target epitopes, which are functionally conserved (present in the majority of currently circulating and reactivated HIV-1 strains) and, at the same time, beneficial (responses to which are associated with better clinical status and control of HIV-1 replication), and rapidly reach protective frequencies upon exposure to the virus. Heterologous prime-boost regimens using virally vectored vaccines are currently the most promising vaccine strategies; nevertheless, induction of robust long-term memory remains challenging. To this end, lentiviral vectors induce high frequencies of memory cells due to their low-inflammatory nature, while typically inducing only low anti-vector immune responses. Here, we describe construction of novel candidate vaccines ZVex.tHIVconsv1 and ZVex.tHIVconsv2, which are based on an integration-deficient lentiviral vector platform with preferential transduction of human dendritic cells and express a bivalent mosaic of conserved-region T cell immunogens with a high global HIV-1 match. Each of the two mosaic vaccines was individually immunogenic. When administered together in heterologous prime-boost regimens with chimpanzee adenovirus and/or poxvirus modified vaccinia virus Ankara (MVA) vaccines to BALB/c and outbred CD1-Swiss mice, they induced a median frequency of over 6,000 T cells/106 splenocytes, which were plurifunctional, broadly specific, and cross-reactive. These results support further development of this vaccine concept.

Innate immune and chemically triggered oxidative stress modifies translational fidelity.

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Vaccinia and influenza A viruses select rather than adjust tRNAs to optimize translation.

Transfer RNAs (tRNAs) are central to protein synthesis and impact translational speed and fidelity by their abundance. Here we examine the extent to which viruses manipulate tRNA populations to favor translation of their own genes. We study two very different viruses: influenza A virus (IAV), a medium-sized (13 kB genome) RNA virus; and vaccinia virus (VV), a large (200 kB genome) DNA virus. We show that the total cellular tRNA population remains unchanged following viral infection, whereas the polysome-associated tRNA population changes dramatically in a virus-specific manner. The changes in polysome-associated tRNA levels reflect the codon usage of viral genes, suggesting the existence of local tRNA pools optimized for viral translation.

Psychiatric complications in Graves' disease.

Background: Mood disorders are common in Graves' disease despite treatment. The pathogenic mechanisms involved are unknown and so is whether previous psychiatric disease influences these symptoms. Methods: This is a longitudinal study conducted in Sweden on 65 women with newly diagnosed Graves' disease and 65 matched controls. Participants were examined during hyperthyroidism and after 15 months of treatment. Examinations included blood sampling, and psychiatric testing with the Comprehensive Psychopathological Rating Scale for Affective Syndromes and the Structured Clinical Interview for DSM-IV - Axis I Disorders. We also performed two analyses of a national population-based registry to determine previous psychiatric diagnoses and previous prescriptions of psychoactive drugs in (i) all patients we asked to participate and (ii) all Swedish women given a diagnosis of hyperthyroidism during 2013-2018, comparing them to matched controls. Results: There was no increased previous psychiatric comorbidity in Graves' patients compared to controls. There was no higher prevalence of psychiatric diagnoses and prescriptions of psychoactive drugs between (i) included GD patients compared to those who declined participation and (ii) women with a hyperthyroidism diagnosis in 5 years prior to their diagnosis, compared to matched controls. Depression scores and anxiety scores were higher in patients compared to controls both during hyperthyroidism (depression (median (IQR): 7.5 (5.0-9.5) vs 1.0 (0.5-2.5) P < 0.001), anxiety: 7.7 (5.0-11) vs 2.5 (1.0-4.0) P < 0.001) and after treatment (depression: 2.5 (1.5-5.0) vs 1.5 (0.5-3.5) P < 0.05), anxiety: 4.0 (2.5-7.5) vs 3.0 (1.5-5.0) P < 0.05). Patients with a previous psychiatric condition, mild eye symptoms, and a younger age had more anxiety at 15 months compared to patients without these symptoms and a higher age (all p<0.05). Conclusion: Graves' disease affects patients' mood despite treatment. A previous psychiatric condition, mild eye symptoms, and a younger age increase the vulnerability for long-lasting symptoms and require specific attention.

A polyvalent RNA vaccine reduces the immune imprinting phenotype in mice and induces neutralizing antibodies against omicron SARS-CoV-2.

Immune imprinting is now evident in COVID-19 vaccinated people. This phenomenon may impair the development of effective neutralizing antibodies against variants of concern (VoCs), mainly Omicron and its subvariants. Consequently, the boost doses with bivalent vaccines have not shown a significant gain of function regarding the neutralization of Omicron. The approach to design COVID-19 vaccines must be revised to improve the effectiveness against VoCs. Here, we took advantage of the self-amplifying characteristic of RepRNA and developed a polyvalent formulation composed of mRNA from five VoCs. LION/RepRNA Polyvalent induced neutralizing antibodies in mice previously immunized with LION/RepRNA D614G and reduced the imprinted phenotype associated with low neutralization capacity of Omicron B.1.1.529 pseudoviruses. The polyvalent vaccine can be a strategy to handle the low neutralization of Omicron VoC, despite booster doses with either monovalent or bivalent vaccines.

RNA replicon delivery via lipid-complexed PRINT protein particles.

Herein we report the development of a nonviral lipid-complexed PRINT (particle replication in nonwetting templates) protein particle system (LPP particle) for RNA replicon delivery with a view toward RNA replicon-based vaccination. Cylindrical bovine serum albumin (BSA) particles (diameter (d) 1 µm, height (h) 1 µm) loaded with RNA replicon and stabilized with a fully reversible disulfide cross-linker were fabricated using PRINT technology. Highly efficient delivery of the particles to Vero cells was achieved by complexing particles with a mixture of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids. Our data suggest that (1) this lipid-complexed protein particle is a promising system for delivery of RNA replicon-based vaccines and (2) it is necessary to use a degradable cross-linker for successful delivery of RNA replicon via protein-based particles.

Clinical trials of self-replicating RNA-based cancer vaccines.

Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma: A Phase 1 Nonrandomized Controlled Trial.

Importance: Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. Objective: To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. Design, Setting, and Participants: This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Interventions: Two doses of IT GLA-SE (5 µg and 10 µg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Main Outcomes and Measures: Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Results: Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. Conclusions and Relevance: In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02180698.

Rendering protein-based particles transiently insoluble for therapeutic applications.

Herein, we report the fabrication of protein (bovine serum albumin, BSA) particles which were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment. Our data demonstrates that cleavage of the cross-linker leaves no chemical residue on the reactive amino group. Delivery of a self-replicating RNA was achieved via the transiently insoluble PRINT protein particles. These protein particles can provide new opportunities for drug and gene delivery.

Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

In vitro and in vivo characterization of microRNA-targeted alphavirus replicon and helper RNAs.

Alphavirus-based replicon vector systems (family Togaviridae) have been developed as expression vectors with demonstrated potential in vaccine development against both infectious diseases and cancer. The single-cycle nature of virus-like replicon particles (VRP), generated by supplying the structural proteins from separate replicable helper RNAs, is an attractive safety component of these systems. MicroRNAs (miRNAs) have emerged as important cellular RNA regulation elements. Recently, miRNAs have been employed as a mechanism to attenuate or restrict cellular tropism of replication-competent viruses, such as oncolytic adenoviruses, vesicular stomatitis virus, and picornaviruses as well as nonreplicating lentiviral and adenoviral vectors. Here, we describe the incorporation of miRNA-specific target sequences into replicable alphavirus helper RNAs that are used in trans to provide the structural proteins required for VRP production. VRP were found to be efficiently produced using miRNA-targeted helper RNAs if miRNA-specific inhibitors were introduced into cells during VRP production. In the absence of such inhibitors, cellular miRNAs were capable of downregulating helper RNA replication in vitro. When miRNA targets were incorporated into a replicon RNA, cellular miRNAs were capable of downregulating replicon RNA replication upon delivery of VRP into animals, demonstrating activity in vivo. These data provide the first example of miRNA-specific repression of alphavirus replicon and helper RNA replication and demonstrate the feasibility of miRNA targeting of expression vector helper functions that are provided in trans.

Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

The Importance of RNA-Based Vaccines in the Fight against COVID-19: An Overview.

In recent years, vaccine development using ribonucleic acid (RNA) has become the most promising and studied approach to produce safe and effective new vaccines, not only for prophylaxis but also as a treatment. The use of messenger RNA (mRNA) as an immunogenic has several advantages to vaccine development compared to other platforms, such as lower coast, the absence of cell cultures, and the possibility to combine different targets. During the COVID-19 pandemic, the use of mRNA as a vaccine became more relevant; two out of the four most widely applied vaccines against COVID-19 in the world are based on this platform. However, even though it presents advantages for vaccine application, mRNA technology faces several pivotal challenges to improve mRNA stability, delivery, and the potential to generate the related protein needed to induce a humoral- and T-cell-mediated immune response. The application of mRNA to vaccine development emerged as a powerful tool to fight against cancer and non-infectious and infectious diseases, for example, and represents a relevant research field for future decades. Based on these advantages, this review emphasizes mRNA and self-amplifying RNA (saRNA) for vaccine development, mainly to fight against COVID-19, together with the challenges related to this approach.

Poxvirus DNA primase.

Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the double-stranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. Furthermore, purified recombinant D5 protein synthesized oligoribonucleotides in vitro. Incorporation of label from [alpha-(32)P]CTP or [alpha-(32)P]UTP into a RNase-sensitive and DNase-resistant product was demonstrated by using single-stranded circular bacteriophage DNA templates and depended on ATP or GTP and a divalent cation. Mutagenesis studies showed that the primase and NTPase activities of the recombinant D5 protein could be independently inactivated. Highly conserved orthologs of D5 are present in all poxviruses that have been sequenced, and more diverged orthologs are found in members of all other families of nucleocytoplasmic large DNA viruses. These viral primases may have roles in initiation of DNA replication or lagging-strand synthesis and represent potential therapeutic targets.