The COVID-19 pandemic is caused by the single-stranded RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus of zoonotic origin that was first detected in Wuhan, China in December 2019. There is evidence that homologous recombination contributed to this cross-species transmission. Since that time the virus has demonstrated a high propensity for human-to-human transmission.Here we report two newly identified adjacent amino acid polymorphisms in the nucleocapsid at positions 203 and 204 (R203K/G204R) due to three adjacent nucleotide changes across the two codons (i.e. AGG GGA to AAA CGA). This new strain within the LGG clade may have arisen by a form of homologous recombination from the core sequence (CS-B) of the transcription-regulating sequences of SAS-CoV-2 itself and has rapidly increased to approximately one third of reported sequences from Europe during the month of March 2020. We note that these polymorphisms are predicted to reduce the binding of an overlying putative HLA-C*07-restricted epitope and that HLA-C*07 is prevalent in Caucasians being carried by >40% of the population. The findings suggest that homologous recombination may have occurred since its introduction into humans and be a mechanism for increased viral fitness and adaptation of SARS-CoV-2 to human populations.

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally.  Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames.  Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host.

Human immunodeficiency virus (HIV) can adapt to an individual’s T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are specific to the host’s human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child’s HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child’s T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs.

Precise clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genetic and epigenetic manipulation of the immune response has become a promising immunotherapeutic approach toward combating tumorigenesis and tumor progression. CRISPR-based immunologic reprograming in cancer therapy comprises the locus-specific enhancement of host immunity, the improvement of tumor immunogenicity, and the suppression of tumor immunoevasion. To date, the ex vivo re-engineering of immune cells directed to inhibit the expression of immune checkpoints or to express synthetic immune receptors (chimeric antigen receptor therapy) has shown success in some settings, such as in the treatment of melanoma, lymphoma, liver, and lung cancer. However, advancements in nuclease-deactivated CRISPR-associated nuclease-9 (dCas9)-mediated transcriptional activation or repression and Cas13-directed gene suppression present novel avenues for the development of tumor immunotherapies. In this review, the basis for development, mechanism of action, and outcomes from recently published Cas9-based clinical trial (genetic editing) and dCas9/Cas13-based pre-clinical (epigenetic editing) data are discussed. Lastly, we review cancer immunotherapy-specific considerations and barriers surrounding use of these approaches in the clinic.