Ethical and scientific considerations
As previously reported in a comparable self-vaccination study by the same human volunteer 9, we consider this to be an ethically and legally legitimate form of experimentation 13. Self- experimentation is a special case of research and limited to individual subjects. It is essential that such self-experimentation does not contravene any questions of personal interest or ethical imperative. Coercion and dependency can be excluded here, ensuring decision-making autonomy. The volunteer in this case is a renowned expert in immunology and thus was able to understand any risks and implications of his own actions. It should therefore be clear that this study was permissible. In addition, relevant precedent exists, where human self- experimentation has opened new avenues for research and contributed to medical progress 14.
We are naturally aware that single case reports cannot provide conclusive evidence or any generalizable results. Rather these findings enable the formulation of new hypotheses. Case reports are attributed with a high sensitivity for detecting novelty and are deemed relevant for medical progress 15. This study is intended as a starting point for intensified discussion and development, rather than a substitute for proper drug development and clinical trials.
Clinical aspects of the vaccination site
As expected with any s.c. vaccination using Montanide 16 or when further adding XS15 as an adjuvant 9, a granuloma developed. It was palpable from day one and grew to a maximum size of approximately 3 x 5 x 1.5 cm by day 12. The skin surface temperature was 37.0°C at the center of the granuloma vs. 36.2°C on adjacent visually unaffected abdominal skin. The granuloma was described as a painless, slightly itchy induration that was sensitive to touch between days 9 and 14, and. It started to shrink in size from day 13.
Ex vivo IFNγ ELISpot results from pre- and post-vaccination PBMCs are shown in Figs 1a & b. The CMV-pp65 HLA-DR epitope YQEFFWDANDIYRIF (amino acids 510–524), which was included as a positive control, was weakly recognized (mean: 16 spots/300,000 cells). Notably, three years prior, vaccination with this peptide resulted in a mean spot count of 525 four weeks after it had been administered, with the same adjuvant and protocol 9. As expected from our previous experience, boosting with one additional vaccination more than one year later with the same peptide (CMV-pp65 510–524) gave a strong response (mean: 910 spots/300,000 cells). The CMV-pp65 HLA-A*01 epitope YSEHPTFTSQY (amino acids 363–373), which had induced a weak response in an ex vivo ELISpot three years prior with a mean spot count of only 12, and showed a negative response before the recent booster vaccination, now gave a mean spot count of 115. These results indicate that a three-year memory against these HLA class II- and class I-restricted CMV-pp65 epitopes prevails after a single vaccination. Importantly, the vaccinated volunteer had previously been tested as CMV seronegative.
No other pre-existing T-cell responses were detectable prior to vaccination. The response for the five predicted SARS-CoV-2 CD8 peptides remained negative after vaccination. Based on previous findings, we speculate that some of these CD8 peptides may show positive results after a 12-day in vitro restimulation (ongoing experiment) or in ex vivo ELISpot with blood obtained later. We intend to test this with blood samples drawn about one month after vaccination.
By contrast, a strong T-cell response was induced against all three SARS-CoV-2 CD4 peptides by a single vaccination: a mean of 214 spots for the nucleoprotein-derived peptide ASAFFGMSRIGMEVT; 71 spots for the nucleoprotein-derived peptide IGYYRRATRRIRGGD; and 29 spots for the envelope protein-derived peptide FYVYSRVKNLNSSRV. Thus, all three SARS- CoV-2-derived CD4 peptides induced T cells to produce IFNγ, most probably representing a TH1 response. Phenotyping of the responding T-cell subsets and the production of further cytokines will be tested using intracellular cytokine staining (ongoing experiments).
Based on these promising results, a second vaccination attempt with a new peptide cocktail designed to be suitable for all individuals independent of their HLA type (see Table 3) was performed by the self-vaccinating individual on April 3, 2020.
The antibodies contained in the plasma of the self-experimenting volunteer and serum of three healthy blood donors were tested using an ELISA. IgG and IgM antibodies against the three SARS-CoV-2 CD4 peptides were not detectable or showed negligible induction (Table 4). We know from previous work that repeated vaccination with CD4 peptides in Montanide with or without additional adjuvants leads to the induction of antibodies 12. We therefore speculate that antibodies against these epitopes may develop later on and will evaluate this with future blood samples. There seems to be a weak recall IgG response against the CMV-pp65 510–524 CD4 epitope YQEFFWDANDIYRIF (an 8-fold increase compared to the reactivity before vaccination). It is interesting that two out of the three healthy donors show high IgG reactivity against YQEFFWDANDIYRIF. Testing serum for antibodies against linear synthetic peptides from all proteins, not only from surmised neutralizing epitope-bearing ones, might be a useful complement to the use of recombinant proteins.
In addition, sera of the vaccinated volunteer and several healthy donors, obtained before the current pandemic, were tested with an ELISA covering SARS-CoV-2 proteins and peptides (Table 5). Further, serum from one donor (HD CoV+-1), was included, who was tested SARS- CoV-2-positive according to medical routines (qRT-PCR) and had recovered from the infection.