The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.

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Over 600 000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers (OPC). A key challenge in understanding HPV immunobiology is the diversity of oncogenic HPV types and the need for multiplexed display of HPV antigens to measure antibody responses. We have generated custom HPV protein microarrays displaying 98 proteins as C-terminal GST fusion proteins, representing eight antigens of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). We demonstrate robust and reproducible protein expression of 96/98 of the antigens using a human cell lysate expression system. The target epitopes and specificities of four monoclonal antibodies were identified. Using sera from ten patients with newly diagnosed OPC and ten controls, we demonstrate specific IgG seroreactivity to HPV16 E1, E2, and E7 (a fold increase of 1.52, 2.19 and 1.35 in cases vs. controls, respectively, all p < 0.005), confirming our prior data on an ELISA platform. We also detect HPV52 E7 Abs in serum from a patient with cervical cancer. The HPV protein array has potential for rapid identification of serologic responses to 12 HPV types.

BackgroundSchistosoma mansoni infection represents a major cause of morbidity and mortality in many areas of the developing world. Effective vaccines against schistosomiasis are not available and disease management relies mainly on treatment with the anthelmintic drug praziquantel. Several promising schistosomal antigens have been evaluated for vaccine efficacy such as Sm14, Sm29 and tetraspanins. However, most investigators examine these promising antigens in animal models individually rather than in properly adjuvanted antigen combinations.MethodsIn the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29. The fusion protein, FSm14/29, was administered to Swiss albino mice either unadjuvanted or adjuvanted with polyinosinic-polycytidylic acid adjuvant, poly(I:C). Mice were challenged with S. mansoni cercariae and different parasitological/immunological parameters were assessed seven weeks post-challenge. Data were analyzed using the ANOVA test with post-hoc Tukey-Kramer test.ResultsMice pre-immunized with unadjuvanted or poly(I:C)-adjuvanted fusion protein showed reduction of adult worm burden of 44.7 and 48.4%, respectively. In addition, significant reduction of tissue egg burdens was observed in mice immunized with the fusion protein when compared with the infected saline/adjuvant negative control groups and groups immunized with the individual Sm14 and Sm29 antigens. Light microscope and scanning electron microscope (SEM) investigation of adult worms recovered from FSm14/29-immunized mice revealed appreciable morphological damage and tegumental deformities. Histopathological examination of liver sections of immunized mice demonstrated reduced granulomatous and inflammatory reactions when compared with infected unvaccinated mice or mice immunized with the individual Sm14 and Sm29 antigens.ConclusionThe findings presented in this study highlight the importance of the fusion protein FSm14/29 as a potential vaccine candidate that is worthy of further investigation.

The application of Cas9 for genetic and epigenetic therapies in humans raises concerns over immunogenicity of this foreign protein. We report pre-existing human CD8+ T cell immunity to Streptococcus pyogenes Cas9 in the majority of healthy individuals screened. In a proof-ofprinciple study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity.Of the Cas9 orthologs derived from bacterial species, the SpCas9 is the best characterized. S. pyogenes is a ubiquitous pathogen, with an annual incidence of 700 million worldwide 26 , but immunity to SpCas9 in humans has not been reported. Here, we sought to characterize the pre-existing immune response to SpCas9 in healthy individuals and to identify the immunodominant T cell epitopes with the aim of developing SpCas9 proteins that have diminished capacity to invoke human adaptive response.CRISPR application for human therapies will span its use both for gene editing (through DNA double-strand breaks) or epigenetic therapies (without DNA double-strand breaks). In fact, recent reports shed light on CRISPR's ability to activate or repress gene expression in mice [27][28][29] , which opens the door to a variety of new therapeutic applications such as activating silent genes, compensating for disrupted genes, cell fate reprogramming, or silencing disrupted genes, without the concern over permanent change in DNA sequence. However, unlike the use of Cas9 for gene editing, which may only require Cas9 presence in cells for a few hours, current techniques for CRISPR-based epigenetic therapies require longer term expression of Cas9 in vivo, possibly for weeks and months 28,29 , which poses the challenge of combating pre-existing immune response towards Cas9. This challenge will need to be addressed before CRISPR application for human therapies, especially for epigenetic therapies, can be fully implemented. Delivery of CRISPR in vivo by incorporating its expression cassette in adeno-associated virus (AAV), will most likely shape many of the initial clinical trials as AAV-based gene delivery is one of the safest and most prevalent forms of gene therapies in human. AAV will enable longer term expression of Cas9, desirable for epigenetic therapies. Therefore, unlike Cas9 delivery in the form of ribonucleoprotein complexes (which are short term), it is highly likely that CRISPR delivery through AAV and its expression within target cells will engage CD8+ T cell immunity.

Background: The most recent (2012) worldwide estimates from International Agency for Research on Cancer indicate that approximately 528,000 new cases and 270,000 deaths per year are attributed to cervical cancer worldwide. The disease is preventable with HPV vaccination and with early detection and treatment of pre-invasive cervical intraepithelial neoplasia, CIN. Antibodies (Abs) to HPV proteins are under investigation as potential biomarkers for early detection.Methods: To detect circulating HPV-specific IgG Abs, we developed programmable protein arrays (NAPPA) that display the proteomes of two low-risk HPV types (HPV6 and 11) and ten oncogenic high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). Arrays were probed with sera from women with CIN 0/I (n=78), CIN II/III (n=84), or invasive cervical cancer (ICC, n=83).Results: Abs to any early (E) HPV protein were detected less frequently in women with CIN 0/I (23.7%) than women with CIN II/III (39.0%) and ICC (46.1%, p<0.04). Of the E Abs, anti-E7 Abs were the most frequently detected (6.6%, 19.5%, and 30.3%, respectively). The least frequently detected Abs were E1 and E2-Abs in CIN 0/I (1.3%) and E1-Abs in CIN II/III (1.2%) and ICC (7.9%). HPV16-specific Abs correlated with HPV16 DNA detected in the cervix in 0% of CIN 0/I, 21.2% of CIN II/III, and 45.5% of ICC. A significant number (29 - 73%) of E4, E7, L1, and L2 Abs had cross-reactivity between HPV types.Conclusion: HPV protein arrays provide a valuable high-throughput tool for measuring the breadth, specificity, and heterogeneity of the serologic response to HPV in cervical disease.

Schistosoma mansoni causes intestinal schistosomiasis, a disease that is prevalent in several regions worldwide. To date, a protective vaccine against S. mansoni is still lacking. Several promising antigens have been discovered and evaluated for vaccine protection, such as Sm14 and Sm28GST. In this short communication, we report the successful detection of an alternatively spliced truncated form of Sm14 which was highly expressed in an Egyptian strain of S. mansoni. This truncated Sm14 (TrSm14) protein was formerly reported to be practically non-existent and its complementary DNA (cDNA) was thought to be 'a rare misprocessing of mRNA precursor'. Our finding demonstrates that there is inter-strain variation in the S. mansoni transcriptome and subsequently in the role/function of the expressed proteins. We expressed TrSm14 successfully in Escherichia coli as a fusion protein with the schistosomal antigen Sm28GST. The fusion protein was purified using metal affinity chromatography and was found to be reactive with serum from S. mansoni-infected patients. This suggests a possible diagnostic value for this protein in detection of anti-schistosomal antibodies. In addition, this fusion protein could offer a potential bivalent vaccine candidate against S. mansoni that is worthy of further investigation.

COVID-19 convalescent plasma (CCP) was one of the first therapeutic options available for the treatment of SARS-CoV-2 infections and continues to be used selectively for immunosuppressed patients. Given the emergence of novel SARS-CoV-2 variants which are resistant to treatment with available monoclonal antibody (MAb) therapy, CCP remains an important therapeutic consideration.