Long-Term Antitumor CD8+ T Cell Immunity Induced by Endogenously Engineered Extracellular Vesicles

Abstract

We developed an innovative method to induce antigen-specific CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This approach employs a DNA vector expressing a mutated HIV-1 Nef protein (Nefmut) deprived of the anti-cellular effects typical of the wild-type isoform, meanwhile showing an unusual efficiency of incorporation into EVs. This function persists even when foreign antigens are fused to its C-terminus. In this way, Nefmut traffics large amounts of antigens fused to it into EVs spontaneously released by the recipient cells. We previously provided evidence that mice injected with a DNA vector expressing the Nefmut/HPV16-E7 fusion protein developed an E7-specific CTL immune response as detected 2 weeks after the second immunization. Here, we extended and optimized the anti-HPV16 CD8+ T cell immune response induced by the endogenously engineered EVs, and evaluated the therapeutic antitumor efficacy over time. We found that the co-injection of DNA vectors expressing Nefmut fused with E6 and E7 generated a stronger anti-HPV16 immune response compared to that observed in mice injected with the single vectors. When HPV16-E6 and -E7 co-expressing tumor cells were implanted before immunization, all mice survived at day 44, whereas no mice injected with either void or Nefmut-expressing vectors survived until day 32 after tumor implantation. A substantial part of immunized mice (7 out of 12) cleared the tumor. When the cured mice were re-challenged with a second tumor cell implantation, none of them developed tumors. Both E6- and E7-specific CD8+ T immunities were still detectable at the end of the observation time. We concluded that the immunity elicited by engineered EVs, besides counteracting and curing already developed tumors, was strong enough to guarantee the resistance to additional tumor attacks. These results can be of relevance for the therapy of both metastatic and relapsing tumors.

Strong SARS-CoV-2 N-specific CD8+ T immunity induced by engineered extracellular vesicles associates with protection from lethal infection in mice

“…Using a viral dose as high as 4.4 LD 50 , we observed protection in mice that had developed more robust N-specific CD8 + T cell immunity. The results in terms of protection from virus challenge seemed to be influenced by some variability in the levels of immunity reached after two injections, similarly to what previously observed in mice immunized by EVs engineered by Nef mut fused with HPV16-E6 and -E7 [12]. Also in this case, mice resisted the tumor implantation carried out before vaccination only in the presence of adequate E6-and E7-specific CD8 + T cell immune responses.…”

“…Engineered EVs can freely circulate into the body, and their internalization into APCs generates a CTL immunity against EV-uploaded antigens in the absence of induction of specific antibodies [17]. The CTL immune response depends on both amounts and efficiency of DNA delivery, lasts several months in peripheral circulation and spleen, and protects mice from both HPV16-and HER2-related cancers [12,13]. The strength of CD8 + T immunity elicited by expression of antigens fused with Nef mut largely exceeds that induced by the expression of foreign antigens alone [12,23].…”

“…The CTL immune response depends on both amounts and efficiency of DNA delivery, lasts several months in peripheral circulation and spleen, and protects mice from both HPV16-and HER2-related cancers [12,13]. The strength of CD8 + T immunity elicited by expression of antigens fused with Nef mut largely exceeds that induced by the expression of foreign antigens alone [12,23].…”

“…Up until now, no vaccine strategy specifically devoted to the induction of CD8 + T immunity has been proposed for humans. To counteract SARS-CoV-2 infection, we applied an original CD8 + T cell-based vaccine platform previously proven to be effective against both HPV16-and HER2-induced cancers [12,13].…”

Virus-Induced CD8+ T-Cell Immunity and Its Exploitation to Contain the SARS-CoV-2 Pandemic

“…Nef mut -based strategy has already been demonstrated to be effective against both transplantable [ 73 ] and ectopic [ 64 ] tumors in view of a strong induction of tumor-specific CTL activity. To apply this technology to a design of anti-SARS-CoV-2 vaccine, DNA vectors expressing the products of fusion between Nef mut and different viral antigens, namely, N- and C-terminal moieties of S (referred to as S1 and S2), M, and N, were generated.…”