Unexplored biosafety issues with respect to self-amplifying (RdRp) RNA-LNP products
Is recombination a potential outcome and if so, we could have a very big problem on our hands: Unanswered questions that need to be addressed
This article is about RNA recombination and the potential dangers of this happening with regard to injecting self-amplifying RNA-LNP injectable products into mammals, including humans. Yesterday I wrote about the incredibly concerning FDA “Study Can Proceed” notification of a Phase I trial of a Biomedical Advanced Research and Development Authority (BARDA)-funded self-amplifying LNP-wrapped ‘pandemic’ H5N1 injectable product in humans. The amount of information pertaining to this trial is anemic at best, and there is no information as to the alphavirus selected, the RNA templates extracted from H5N1 used as inserts, or the LNP formulation. I would assume it’s the same as for the modified mRNA-LNP COVID-19 injectable products but I am not certain.
I conclude this article with 5 exploratory questions answered by Grok AI.
Arcturus is actively engaged with the U.S. government to prepare for the next pandemic.1
If that sentence doesn’t concern you, it should.
ARCT-2304 is a sa-mRNA vaccine candidate formulated within a lipid nanoparticle (LNP). The sa-mRNA vaccine candidate is designed to make many copies of mRNA within the host cell after intramuscular injection to achieve enhanced expression of haemagglutinin (HA) and neuraminidase (NA) antigens, thereby enabling lower doses than conventional mRNA vaccines.
The Phase 1 clinical trial is designed to evaluate the safety, reactogenicity, and immunogenicity of ARCT-2304 as a potential vaccine to protect against the highly pathogenic H5N1 avian influenza.2
The product in Phase I trials is called LUNAR-FLU (ARCT-2304) Pandemic for ‘Pandemic Influenza’ (H5N1). You can read about that here.
Influenza A virus subtype H5N1 (A/H5N1) is a subtype of the influenza A virus, which causes influenza (flu), predominantly in birds, but can infect humans.3
The two glycoproteins embedded in the influenza viral membrane are hemagglutinin (HA) and neuraminidase (NA).4 The HA attaches to cell receptors and plays an important role in the release of the viral RNA into the cell via viral and cellular membrane fusion. HA has to be cleaved by cellular proteases to be active as a fusion protein.5 NA plays a crucial role in the virus’s life cycle by helping to release newly formed virions from infected cells.6
Venezuelan equine encephalitis virus (VEEV)
VEEV is a mosquito-borne viral pathogen that causes Venezuelan equine encephalitis or encephalomyelitis (VEE). VEE can affect all equine species, such as horses, donkeys, and zebras. After infection, equines may suddenly die or show progressive central nervous system disorders. Humans also can contract this disease. Healthy adults who become infected by the virus may experience flu-like symptoms, such as high fevers and headaches. People with weakened immune systems and the young and the elderly can become severely ill or die from this disease.7
Recombination
Recombination can be replicative or non-replicative. This means that recombination can occur during the replication process or can involve the direct joining of pre-existing RNA molecules. In the case of the former, the viral RNA-dependent RNA polymerase (RdRp) - responsible for synthesizing new RNA strands - can switch templates, incorporating sequences from one viral RNA molecule into another (donor vs. acceptor) resulting in the creation of a new recombinant RNA molecule.8 In the case of the latter, there is no involvement of RdRp - two RNA molecules are joined at specific sites.
VEEV is an unsegmented RNA virus - this means instead of having their genome divided into multiple independent RNA segments where each segment encodes one or more viral protein, they have a continuous RNA strand that contains all the genes necessary for the virus to replicate and produce new viral particles. A recombination event between two unsegmented RNA viruses is demonstrated below. VEEV carries the RdRp gene. The RdRp gene is essential to viral replication and production of new virions in non-segmented RNA viruses. It’s also notable that recombination can occur more frequently in non-segmented RNA viruses because of their continuous genomes.
So since RdRp synthesizes new RNA strands by copying a template RNA strand, a recombination event can occur in the presence of another compatible RNA template within the same host cell, say, exogenously introduced foreign RNA encoding spike or NA/HA from influenza. When the genetic material is replicated, RdRp may switch between these templates, incorporating segments from each into the new RNA strand. This would result in a new viral genome containing a mixture of genetic material from the parent virus and the foreign RNA material. RdRp is also error-prone so can contribute to recombination events from this vantage point as well.
It is also notable that there will be compatibility issues between viruses with regard to expression of surface proteins, for example. The compatibility between the alphavirus that they chose to use for their self-amplifying products (it could be VEEV), and the chunks of genetic material loaded into the sub-genomic-material-deleted VEEV template, downstream of RdRp gene - be it spike from SARS-2 or HA/NA from influenza, are potentially low, but we don’t really know. It will depend on how (and if) exactly they modified the inserts. Depending on structural and functional compatibility between VEEV's alphavirus envelope and influenza's envelope proteins, the result of the recombination might not be a stable, functional virus; it might instead lead to either non-viable recombinants or viruses with reduced fitness. We can only hope, just like Birx told us to.
More specifically, the lipid compositions and protein anchoring abilities of the alphavirus would have to be compatible with whatever protein was available for expression - be it spike or HA/NA. The genes for the spike/flu proteins would need to be expressed correctly in the context of VEEV's replication cycle to be processed and transported to the membrane, and the glycosylation patterns of VEEV proteins versus spike/flu proteins might differ as well, potentially impacting compatibility.
The thing that bothers me the most is the proprietary excuses that these pharma companies cling to. If we can’t know what the sequences for the alphavirus template are, or what the inserted genes (be it spike or HA/NA) are exactly, how can we assess compatibility issues with regard to potential recombination concerns? Were these guys codon-optimized? Apparently for the KOSTAIVE® (ARCT-154) Monovalent: JN.1 product, the spike gene was codon optimized. We know nothing about this Phase trial LUNAR®-FLU (ARCT-2304) Pandemic self-amplifying flu product paid for by DARPA.
I asked Grok the following 5 questions:
What would happen to a Venezuelan equine encephalitis virus if it recombined with a self-amplifying gene therapy product with H5N1 hemagglutinin and neuraminidase genes?
Answer
If a Venezuelan equine encephalitis virus (VEEV) were to recombine with a self-amplifying gene therapy product incorporating genes for H5N1 hemagglutinin (HA) and neuraminidase (NA), several potential outcomes could be hypothesized:
Increased Pathogenicity or Virulence:
Enhanced Cell Entry: The HA gene from H5N1 could potentially enhance the ability of the virus to enter host cells, given that HA is crucial for the attachment and entry of influenza viruses into host cells. If this gene were to be expressed on the surface of a VEEV-like particle, it might allow the virus to infect cells that might not typically be susceptible to VEEV alone.
Altered Tropism: The presence of H5N1 genes might change the tissue tropism of the virus, possibly leading it to infect different types of cells or tissues than the original VEEV. This could potentially increase the spread or severity of infection within a host.
Immune Response Modulation:
Antigen Presentation: The inclusion of H5N1 HA and NA antigens might trigger an immune response typical of influenza, potentially confusing or overloading the immune system, especially if it encounters both viral proteins in a novel context. This could lead to an altered immune response where the body might be less effective at neutralizing the virus due to the presence of unexpected antigens.
Replication Dynamics:
Self-Amplification: If the gene therapy product uses a self-amplifying RNA system, this could potentially increase the production of viral proteins or RNA, leading to higher viral loads within infected cells. This might result in a more robust infection cycle, potentially increasing the cytopathic effect on host cells.
Potential for New Vaccine or Therapeutic Development:
While this recombination could pose risks, it also presents an opportunity for developing novel vaccines or therapeutic strategies. For instance, using VEEV as a vector for delivering influenza antigens could lead to a highly immunogenic chimeric virus that could be used in vaccine development, although this would come with significant biosafety considerations.
Biosafety Concerns:
Risk of Generating a New Pathogen: Such a chimeric virus might possess characteristics not found in either parent virus, potentially creating a new pathogen with unpredictable behavior. This would raise significant biosafety issues, as the new virus might have increased transmissibility or pathogenicity.
Regulatory and Ethical Issues:
Any work involving the creation or study of such recombinant viruses would face intense scrutiny from regulatory bodies due to the potential biosecurity and biosafety risks. Ethical considerations around manipulating viruses to potentially increase their pathogenicity would also be paramount.
Given these points, the exact outcome would depend heavily on how these genetic elements interact, the stability of the recombinant genome, and the host's immune response. Such a scenario underscores the importance of stringent controls in genetic engineering and virology to prevent unintended consequences like pandemics or ecological disruptions.
If a person was infected with Venezuelan equine encephalitis virus and they got injected with a self-amplifying flu vaccine that had genes encoding hemagglutinin and neuraminidase, could a recombination event ensue?
Answer
The possibility of a recombination event between a Venezuelan equine encephalitis virus (VEEV) infection and a self-amplifying RNA (saRNA) vaccine expressing influenza virus hemagglutinin (HA) and neuraminidase (NA) genes involves several complex biological processes:
Co-infection Requirement:
For recombination to occur, both viruses would need to infect the same cell at the same time. With VEEV, an alphavirus, primarily infecting neural and lymphatic tissues, and an influenza-based vaccine potentially targeting respiratory cells or inducing expression in muscle or skin cells if administered intramuscularly or intradermally, the likelihood of co-infection in the same cell type might be low. However, if the vaccine's RNA is capable of systemic distribution or if there's a chance of cross-infection in shared cell types, this risk increases.
RNA Recombination:
RNA viruses like VEEV and influenza viruses have RNA genomes that can undergo recombination, although this is more common among related viruses. VEEV is an alphavirus, and influenza viruses belong to the orthomyxovirus family, making natural recombination between these two less likely due to their different genetic architectures. However, human intervention through genetic engineering could potentially facilitate such events.
Mechanism of Recombination:
If the self-amplifying vaccine uses RNA, there would need to be a mechanism for the RNA to enter the same replication compartment as VEEV for recombination to occur. This could theoretically happen via:
Homologous Recombination: Where similar RNA sequences align and exchange genetic material. However, this would require sufficient sequence homology between VEEV and the influenza genes, which is unlikely given their different origins.
Non-homologous Recombination: Less sequence-specific but less common, where RNA segments could be joined together through mechanisms like template switching during replication.
Genetic Compatibility:
The expression of influenza HA and NA in a VEEV backbone would face challenges due to codon usage differences, protein folding requirements, and the need for post-translational modifications specific to each virus's lifecycle.
Safety Features of saRNA Vaccines:
Modern saRNA vaccines often include safety features like:
Deletion of Essential Genes: Removing genes crucial for replication (like the capsid gene in VEEV-based vectors) in one or both viruses to prevent full virus particle formation.
Use of Split Helper Systems: Separating necessary genes onto different RNA molecules to reduce the likelihood of generating replication-competent virus through recombination.
Implications for Humans:
If such a recombination event did occur, it would likely lead to an attenuated or non-viable virus due to the genetic mismatch. However, in an unlikely scenario where a viable recombinant emerged, it could potentially display:
Altered Pathogenicity: Depending on how the genes function together.
New Antigenic Properties: Possibly eliciting an immune response against influenza or VEEV, or both, in novel ways.
Given all these considerations, while not entirely impossible, the probability of a functional recombination event happening in a human host after vaccination, leading to a viable and pathogenic chimeric virus, is low due to biological barriers, the design of modern vaccines, and the lack of extensive genetic homology. However, this scenario would be of great concern in terms of biosafety, leading to rigorous testing and safety protocols in the development of such vaccines.
If a person was infected with Venezuelan equine encephalitis virus and they got injected with a self-amplifying vaccine that had genes encoding sars-2 spike, could a recombination event ensue?
Answer
The scenario where a person infected with Venezuelan equine encephalitis virus (VEEV) receives an injection of a self-amplifying RNA (saRNA) vaccine expressing the SARS-CoV-2 spike protein introduces several considerations for potential recombination:
Co-infection and Cellular Context:
Recombination typically requires that both viral RNAs are present in the same cell at the same time. For VEEV, which can infect neural and lymphatic tissues, and an saRNA vaccine expressing SARS-CoV-2 spike, which might be targeted to express primarily in muscle cells or skin cells where it's injected, co-infection in the same cell type might not be straightforward. However, VEEV's broader tissue tropism compared to influenza could increase this possibility.
RNA Recombination Mechanisms:
RNA viruses can undergo recombination through:
Homologous Recombination: This would require some sequence similarity between VEEV RNA and the SARS-CoV-2 spike mRNA, which is unlikely as these viruses are from different families (Togaviridae for VEEV and Coronaviridae for SARS-CoV-2).
Non-homologous Recombination: More likely but still rare. RNA polymerases can occasionally switch templates during replication, but this process is stochastic and less common between such divergent viruses.
Genetic Compatibility:
The SARS-CoV-2 spike protein would need to be compatible with VEEV's replication machinery and structural proteins if a functional recombinant virus were to arise. This would be highly improbable due to:
Different Codon Usage: The codon usage optimized for human cells in the vaccine might not match the requirements of VEEV's RNA replication machinery.
Structural Incompatibility: The spike protein's requirement for trimerization and specific host cell interactions might not be fulfilled by the VEEV envelope.
Potential Outcomes:
Non-viable Recombinants: Most recombination events would likely result in defective viruses incapable of replication or infectivity.
Attenuated Virus: If by chance, a recombination event did produce a viable chimeric virus, it might be attenuated because the SARS-CoV-2 spike might interfere with normal VEEV functions or vice versa.
Novel Pathogen: Though extremely unlikely, if the recombination led to a functional virus with traits from both, this could theoretically result in a new pathogen with unpredictable characteristics. However, this would require overcoming significant genetic and structural hurdles.
Safety Measures in Vaccine Design:
Self-amplifying RNA vaccines usually incorporate safety features:
Non-infectious Delivery: The RNA is typically encapsulated to prevent premature degradation or off-target effects.
Split Helper Constructs: Some designs split the helper RNAs, making it less likely for a complete, infectious virus to be produced through recombination.
Lack of Structural Proteins: Often, structural proteins necessary for assembly into infectious particles are not included in the vaccine to prevent this scenario.
Given the genetic dissimilarity between VEEV and SARS-CoV-2, along with the design of self-amplifying vaccines to prevent such events, the likelihood of a recombination leading to a new, viable, and pathogenic virus is considered very low. However, such possibilities underscore the importance of rigorous biosafety testing in vaccine development to ensure that even unlikely scenarios are accounted for.
If a person was infected with an alphavirus and they got injected with a self-amplifying vaccine that had genes encoding SARS-2 spike, could a recombination event ensue?
Answer
The possibility of a recombination event between an alphavirus infection and a self-amplifying RNA (saRNA) vaccine encoding the SARS-CoV-2 spike protein involves several factors:
Co-infection:
For recombination to occur, both the alphavirus RNA and the saRNA from the vaccine would need to be present in the same cell simultaneously. While this is conceivable, especially if the vaccine's RNA has some degree of systemic distribution after injection, the likelihood still depends on the tropism of the alphavirus and where the vaccine is administered.
Mechanisms of Recombination:
Homologous Recombination:
This is less likely due to the genetic dissimilarity between alphaviruses and SARS-CoV-2. They belong to different families with little expected sequence homology.
Non-homologous Recombination:
Although less common, non-homologous recombination could theoretically occur if the RNA polymerases from both viruses interact in a way that allows for RNA strand switching. However, this would be a rare event due to the distinct replication mechanisms and RNA structures.
Genetic and Structural Barriers:
Codon Usage: The codon optimization for human expression in the vaccine might differ from the alphavirus's codon bias, complicating any functional integration into the alphavirus genome.
Protein Functionality: The SARS-CoV-2 spike protein has specific structural and functional requirements for its trimeric form and interaction with the human ACE2 receptor, which would not naturally integrate into the alphavirus lifecycle or envelope.
Alphavirus Replication Cycle:
Alphaviruses like VEEV replicate in the cytoplasm using their own replicase. For a recombination event to occur, the SARS-CoV-2 spike RNA would need to be recognized by this replicase or somehow influence the replication process.
Safety Features of saRNA Vaccines:
Modern saRNA vaccines often include:
Non-infectious Delivery: Encapsulation in lipid nanoparticles or other carriers that limit the RNA's ability to spread beyond the injection site or targeted cells.
Lack of Structural Proteins: The vaccine usually does not include alphavirus structural proteins necessary for forming infectious particles, reducing the likelihood of recombination leading to a functional virus.
Outcome of Hypothetical Recombination:
If recombination did occur, it's most likely that the resulting RNA would be non-viable or produce an attenuated virus because:
The SARS-CoV-2 spike would likely not be compatible with the alphavirus envelope structure.
Even if a chimeric virus was formed, it might not efficiently replicate or infect cells due to mismatched protein interactions.
Biosafety Considerations:
While the chances are low, the potential for such events is taken seriously. Therefore, saRNA vaccines are designed with safety in mind, and extensive testing is conducted to ensure no recombination with related or unrelated viruses can produce a new pathogenic entity.
Given these considerations, the probability of creating a functional, pathogenic virus through recombination between an alphavirus and an saRNA vaccine expressing SARS-CoV-2 spike is considered extremely low due to the biological and genetic barriers. However, this scenario highlights the importance of biosafety, biosecurity, and thorough testing protocols in the development and administration of RNA-based vaccines.
If a person was infected with an alphavirus and they got injected with a self-amplifying flu vaccine that had genes encoding hemagglutinin and neuraminidase, could a recombination event ensue?
Answer
The scenario where an individual infected with an alphavirus receives a self-amplifying RNA (saRNA) vaccine targeting influenza A viruses, which encodes hemagglutinin (HA) and neuraminidase (NA), involves several key considerations for the potential of a recombination event:
Co-infection:
For recombination to happen, both the alphavirus RNA and the influenza-specific saRNA must be present within the same cell at the same time. Alphaviruses like VEEV predominantly infect neural and lymphatic tissues, which might not overlap significantly with the primary sites of influenza virus infection or where a vaccine would induce protein expression (usually muscle or skin if injected intramuscularly or intradermally).
Mechanisms of Recombination:
Homologous Recombination: Influenza and alphaviruses are from different viral families with distinct genomic structures, making homologous recombination less likely due to insufficient sequence similarity.
Non-homologous Recombination: While more feasible, this type of recombination would still be an unusual event, particularly given the different replication strategies of these viruses.
RNA Replication and Compatibility:
Alphaviruses replicate their RNA in the cytoplasm using their own RNA-dependent RNA polymerase, whereas influenza viruses replicate in the nucleus. The machinery for replication is very different:
The influenza HA and NA genes would need to interact or be incorporated into the replication process of an alphavirus, which typically does not include processing in the nucleus.
Structural and Functional Compatibility:
Even if recombination occurred, the incorporation of influenza HA and NA into an alphavirus particle would face significant challenges:
Structural Incompatibility: The HA and NA proteins are structurally adapted to the influenza virus envelope, not the alphavirus envelope.
Functionality: HA and NA have specific roles in influenza virus entry and release that might not be compatible with the lifecycle of an alphavirus.
Vaccine Design Features:
Modern saRNA vaccines are designed to minimize such risks:
Non-infectious: The RNA is encapsulated in delivery systems like lipid nanoparticles, which target the RNA to specific cell types, reducing the chance of it reaching areas where alphaviruses replicate.
Split Helper Systems: Some vaccines split the genes for RNA replication from those encoding antigens, making it more difficult for a complete, infectious virus to emerge through recombination.
Potential Outcomes:
If recombination did occur, it would likely result in:
Non-functional Viruses: Most recombinants would be non-viable or incapable of causing disease because of the genetic and structural mismatches.
Attenuated Virus: In the rare event of a functional recombinant, it might exhibit reduced virulence due to the disruption of normal viral function by the foreign genes.
Biosafety and Monitoring:
Vaccine development includes extensive safety testing to ensure that even theoretical risks like recombination are minimized. Continuous monitoring for unexpected viral variants would also be part of post-licensure surveillance.
Given these points, while the concept of recombination is theoretically possible, the practical likelihood of creating a viable, pathogenic virus from such an event is low due to the significant genetic, structural, and functional barriers between alphaviruses and influenza viruses. However, this scenario underscores the importance of careful design, testing, and monitoring in vaccine development to address even the most remote possibilities.
https://www.businesswire.com/news/home/20241111138781/en/Arcturus-Therapeutics-Receives-Clearance-from-FDA-to-Begin-H5N1-Pandemic-Flu-Vaccine-Clinical-Trial
https://www.businesswire.com/news/home/20241111138781/en/Arcturus-Therapeutics-Receives-Clearance-from-FDA-to-Begin-H5N1-Pandemic-Flu-Vaccine-Clinical-Trial
https://en.wikipedia.org/wiki/H5N1_genetic_structure
https://virology.ws/2009/07/28/influenza-neuraminidase-and-h5n1-pathogenicity/
https://virology.ws/2009/06/22/influenza-ha-cleavage-is-required-for-infectivity/
https://en.wikipedia.org/wiki/H5N1_genetic_structure
https://en.wikipedia.org/wiki/Venezuelan_equine_encephalitis_virus
Simon-Loriere, E., Holmes, E. Why do RNA viruses recombine?. Nat Rev Microbiol 9, 617–626 (2011). https://doi.org/10.1038/nrmicro2614
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'Unexplored' is a good way of putting it, kind of like the 'unexplored' consequences of hunting blindfolded!