SARS-CoV-2 Vaccines: Inactivation by Gamma Irradiation for T and B Cell Immunity.

Despite accumulating preclinical data demonstrating a crucial role of cytotoxic T cell immunity during viral infections, ongoing efforts on developing COVID-19 vaccines are mostly focused on antibodies. In this commentary article, we discuss potential benefits of cytotoxic T cells in providing long-term protection against COVID-19. Further, we propose that gamma-ray irradiation, which is a previously tested inactivation method, may be utilized to prepare an experimental COVID-19 vaccine that can provide balanced immunity involving both B and T cells.

A pro-survival role for the intracellular granzyme B inhibitor Serpinb9 in natural killer cells during poxvirus infection.

Intracellular serpins are proposed to inactivate proteases released from lysosome-related organelles into the host cell interior, preventing cell death. Serpinb9 opposes the immune cytotoxic protease, granzyme B, and in a number of settings protects cells against granzyme B-mediated cell death. Using a knockout mouse line engineered to express green fluorescent protein under the serpbinb9 promoter, we demonstrate that serpinb9 is vital for host survival during Ectromelia virus infection by maintaining both mature natural killer NK) cells, and activated CD8+ T cells. Serpinb9 expression parallels granzyme B expression within both populations during infection. Maturing serpinb9-null NK cells exhibit higher levels of granzyme B-mediated apoptosis during infection; hence there are fewer mature NK cells, and these cells also have lower cytotoxic potential. Thus the serpinb9-granzyme B axis is important for homeostasis of both major cytotoxic effector cell populations.

Ectromelia virus N1L is essential for virulence but not dissemination in a classical model of mousepox.

Mousepox is caused by the orthopoxvirus ectromelia virus (ECTV), and is thought to be transmitted via skin abrasions. We studied the ECTV virulence factor N1 following subcutaneous infection of mousepox-susceptible BALB/c mice. In this model, ECTV lacking N1L gene was attenuated more than 1000-fold compared with wild-type virus and replication was profoundly reduced as early as four days after infection. However, in contrast to data from an intranasal model, N1 protein was not required for virus dissemination. Further, neither T cell nor cytokine responses were enhanced in the absence of N1. Together with the early timing of reduced virus titres, this suggests that in a cutaneous model, N1 exerts its function at the level of infected cells or in the inhibition of the very earliest effectors of innate immunity.

MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies.

MHCII molecules are heterodimeric cell surface proteins composed of an α and ß chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAß(-/-) (I-ß(-/-)) and the double knockout (I-Aαxß(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-ß(-/-) or I-Aαxß(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

Bis(methyl)gliotoxin proves to be a more stable and reliable marker for invasive aspergillosis than gliotoxin and suitable for use in diagnosis.

The virulence factor gliotoxin (GT) and its inactive derivative, bis(methylthio)gliotoxin (bmGT), are produced by pathogens of the genus Aspergillus. Here we report the detection of GT and bmGT in serum of humans at risk of invasive aspergillosis (IA) as well as in cultures of fungal isolates derived from patients with proven infection with A. fumigatus. Although both compounds are readily recoverable from spiked human serum or plasma, only bmGT is retained in whole blood, indicating that bmGT may be the better marker for in vivo detection. Accordingly, bmGT was found more frequently than GT in samples from patients at risk of IA and incultures of clinical isolates of A. fumigatus. In some cases, bmGT was detected before mycologic evidence ofinfection was gained. Importantly, neither GT nor bmGT was found in serum from healthy donors or from neutropenic patients without any sign of infection. Thus, bmGT presence might provide a more reliable indicator of A. fumigatus infections than GT. Due to its simplicity and sensitivity, a diagnostic technology based on this test could be easily adopted in clinical laboratories to help in the diagnosis of this often fatal fungal infection.

Gamma-irradiated influenza virus uniquely induces IFN-I mediated lymphocyte activation independent of the TLR7/MyD88 pathway.

BACKGROUND: We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that γ-irradiated SFV does not induce IFN-I and the associated lymphocyte activation. PRINCIPAL FINDINGS: Here we report that, in contrast to SFV, γ-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only γ-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways. CONCLUSIONS: Our data suggest an important mechanism for the recognition of γ-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of γ-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.

The ectromelia virus SPI-2 protein causes lethal mousepox by preventing NK cell responses.

Ectromelia virus (ECTV) is a natural pathogen of mice that causes mousepox, and many of its genes have been implicated in the modulation of host immune responses. Serine protease inhibitor 2 (SPI-2) is one of these putative ECTV host response modifier proteins. SPI-2 is conserved across orthopoxviruses, but results defining its mechanism of action and in vivo function are lacking or contradictory. We studied the role of SPI-2 in mousepox by deleting the SPI-2 gene or its serine protease inhibitor reactive site. We found that SPI-2 does not affect viral replication or cell-intrinsic apoptosis pathways, since mutant viruses replicate in vitro as efficiently as wild-type virus. However, in the absence of SPI-2 protein, ECTV is attenuated in mousepox-susceptible mice, resulting in lower viral loads in the liver, decreased spleen pathology, and substantially improved host survival. This attenuation correlates with more effective immune responses in the absence of SPI-2, including an earlier serum gamma interferon (IFN-γ) response, raised serum interleukin 18 (IL-18), increased numbers of granzyme B(+) CD8(+) T cells, and, most notably, increased numbers and activation of NK cells. Both virus attenuation and the improved immune responses associated with SPI-2 deletion from ECTV are lost when mice are depleted of NK cells. Consequently, SPI-2 renders mousepox lethal in susceptible strains by preventing protective NK cell defenses.

In vivo elimination of MHC-I-deficient lymphocytes by activated natural killer cells is independent of granzymes A and B.

NK cells kill target cells mainly via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and induction of apoptosis is severely impaired in the absence of gzm A and B. However, their importance for in vivo cytotoxicity by cytotoxic T cells has been questioned. We used an in vivo NK cytotoxicity assay, in which splenocytes from wild-type and ß(2)microglobulin-deficient (MHC-I(neg)) mice are co-injected into recipients whose NK cells were activated by virus infection or synthetic Toll-like receptor ligands. Elimination of adoptively transferred MHC-I(neg) splenocytes was unimpaired in the absence of gzmA and gzmB, but dependent on perforin. This target cell rejection was NK cell dependent, since NK cell depletion abrogated it. Furthermore, target cell elimination in vivo was equally rapid in both wild-type and gzmAxB-deficient recipients, with the majority of specific target cells lost from lymphoid tissue within less than one to two hours after transfer. Thus, similar to T cell cytotoxicity, the contribution of gzmA and B to in vivo target cell elimination remains unresolved.

TCR down-regulation boosts T-cell-mediated cytotoxicity and protection against poxvirus infections.

Cytotoxic T (Tc) cells play a key role in the defense against virus infections. Tc cells recognize infected cells via the T-cell receptor (TCR) and subsequently kill the target cells by one or more cytotoxic mechanisms. Induction of the cytotoxic mechanisms is finely tuned by the activation signals from the TCR. To determine whether TCR down-regulation affects the cytotoxicity of Tc cells, we studied TCR down-regulation-deficient CD3γLLAA mice. We found that Tc cells from CD3γLLAA mice have reduced cytotoxicity due to a specific deficiency in exocytosis of lytic granules. To determine whether this defect was reflected in an increased susceptibility to virus infections, we studied the course of ectromelia virus (ECTV) infection. We found that the susceptibility to ECTV infection was significantly increased in CD3γLLAA mice with a mortality rate almost as high as in granzyme B knock-out mice. Finally, we found that TCR signaling in CD3γLLAA Tc cells caused highly increased tyrosine phosphorylation and activation of the c-Cbl ubiquitin ligase, and that the impaired exocytosis of lytic granules could be rescued by the knockdown of c-Cbl. Thus, our work demonstrates that TCR down-regulation critically increases Tc cell cytotoxicity and protection against poxvirus infection.

Antigen-specific activation thresholds of CD8+ T cells are independent of IFN-I-mediated partial lymphocyte activation.

Type-I IFN (IFN-I) are highly pleiotropic cytokines known to modulate immune responses and play an early central role in mediating antiviral defenses. We have shown that IFN-I mediate transient up-regulation of a distinct subset of lymphocyte surface activation markers on both B and T cells in vivo independent of cognate antigen: a state referred to as 'partial lymphocyte activation'. Here we investigated in vitro the possibility that partial lymphocyte activation may serve to lower the antigen-specific activation thresholds for T cells. We found that the kinetics of Ca(2+) flux in T cells responding to TCR cross-linking was not enhanced in partially activated T cells. Furthermore, following TCR stimulation with anti-cluster of differentiation (CD) 3 epsilon, a lower proportion of partially activated than naive T cells proliferated. In contrast, the proliferation of partially activated and naive ovalbumin peptide (OVAp, SIINFEKL) specific CD8(+) T cells (OT-I CD8(+) T cells) was similar when stimulated with OVAp. Surprisingly, using an enzyme-linked immunospot (ELISPOT) assay for IFN-gamma secretion, we found that a higher number of partially activated OT-I CD8(+) T cells expressed effector functions than did naive OT-I CD8(+) T cells. This is most readily explained by an increased survival of activated antigen-specific CD8(+) T cells from a pool of partially activated T cells than naive T cells. Overall, when examining the effects of early (Ca(2+) flux), intermediate (proliferation) or late events (IFN-gamma secretion) of T-cell activation, we found that partial activation promotes the survival but does not alter the antigen-specific activation thresholds of CD8(+) T cells.

Effect of inactivation method on the cross-protective immunity induced by whole 'killed' influenza A viruses and commercial vaccine preparations.

We have recently shown that intranasal (i.n.) administration of gamma-irradiated A/PR/8 [A/Puerto Rico/8/34 (H1N1)] protects mice against lethal avian influenza A/Vietnam/1203/2004 (H5N1) and other heterosubtypic influenza A infections. Here, we used gamma-irradiated, formalin- and UV-inactivated A/PC [A/Port Chalmers/1/73 (H3N2)] virus preparations and compared their ability to induce both homologous and heterosubtypic protective immunity. Our data show that, in contrast to i.n. vaccination with formalin- or UV-inactivated virus, or the present commercially available trivalent influenza vaccine, a single dose of gamma-ray-inactivated A/PC (gamma-A/PC) conferred significant protection in mice against both homologous and heterosubtypic virus challenges. A multiple immunization regime was required for formalin-inactivated virus preparations to induce protective immunity against a homotypic virus challenge, but did not induce influenza A strain cross-protective immunity. The highly immunogenic gamma-A/PC, but not formalin- or UV-inactivated A/PC, nor the currently available subvirion vaccine, elicited cytotoxic T-cell responses that are most likely responsible for the cross-protective and long-lasting immunity against highly lethal influenza A infections in mice. Finally, freeze-drying of gamma-A/PC did not affect the ability to induce cross-protective immunity.

Cytotoxic T cells are the predominant players providing cross-protective immunity induced by {gamma}-irradiated influenza A viruses.

We previously demonstrated that a single dose of nonadjuvanted intranasal gamma-irradiated influenza A virus can provide robust protection in mice against both homologous and heterosubtypic challenges, including challenge with an H5N1 avian virus strain. We investigated the mechanism behind the observed cross-protection to define which arms of the adaptive immune response are involved in mediating this protection. Studies with gene knockout mice showed the cross-protective immunity to be mediated mainly by T cells and to be dependent on the cytolytic effector molecule perforin. Adoptive transfer of memory T cells from immunized mice, but not of memory B cells, protected naïve recipients against lethal heterosubtypic influenza virus challenge. Furthermore, gamma-irradiated influenza viruses induced cross-reactive Tc-cell responses but not cross-neutralizing or cross-protective antibodies. In addition, histological analysis showed reduced lung inflammation in vaccinated mice compared to that in unvaccinated controls following heterosubtypic challenge. This reduced inflammation was associated with enhanced early recruitment of T cells, both CD4(+) and CD8(+), and with early influenza virus-specific cytotoxic T-cell responses. Therefore, cross-protective immunity induced by vaccination with gamma-irradiated influenza A virus is mediated mainly by Tc-cell responses.

Caspase-dependent inhibition of mousepox replication by gzmB.

BACKGROUND: Ectromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants. METHODS AND FINDINGS: Here we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB(+) Tc cells) or granzyme A and granzyme B deficient (GzmAxB(-/-) Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers. SIGNIFICANCE: Out findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.

Cutting edge: rapid and efficient in vivo cytotoxicity by cytotoxic T cells is independent of granzymes A and B.

Cytotoxic T (Tc) cells lyse target cells via exocytosis of granules containing perforin (perf) and granzymes (gzm). In vitro, gzm delivery into the target cell cytosol results in apoptosis, and in the absence of gzm A and B the induction of apoptosis is severely impaired. However, using in vivo Tc cell killing assays, we find that virus-immune, gzm A x B-deficient (gzmAxB(-/-)) mice are competent to eliminate adoptively transferred target cells pulsed with an immunodominant Tc cell determinant as rapidly and completely as their wild-type counterparts. Specific target cell elimination occurred with similar kinetics in both spleen and lymph nodes. Thus, neither gzmA nor gzmB are required for rapid and efficient in vivo cytotoxicity by Tc cells.

Intranasal flu vaccine protective against seasonal and H5N1 avian influenza infections.

BACKGROUND: Influenza A (flu) virus causes significant morbidity and mortality worldwide, and current vaccines require annual updating to protect against the rapidly arising antigenic variations due to antigenic shift and drift. In fact, current subunit or split flu vaccines rely exclusively on antibody responses for protection and do not induce cytotoxic T (Tc) cell responses, which are broadly cross-reactive between virus strains. We have previously reported that gamma-ray inactivated flu virus can induce cross-reactive Tc cell responses. METHODOLOGY/PRINCIPAL FINDING: Here, we report that intranasal administration of purified gamma-ray inactivated human influenza A virus preparations (gamma-Flu) effectively induces heterotypic and cross-protective immunity. A single intranasal administration of gamma-A/PR8[H1N1] protects mice against lethal H5N1 and other heterotypic infections. CONCLUSIONS/SIGNIFICANCE: Intranasal gamma-Flu represents a unique approach for a cross-protective vaccine against both seasonal as well as possible future pandemic influenza A virus infections.

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation.

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.

Restricted Semliki Forest virus replication in perforin and Fas-ligand double-deficient mice.

Previously, we have shown that mice defective in granule exocytosis and/or Fas.L/Fas-mediated cytolytic pathways are significantly more resistant to alphavirus, Semliki Forest virus (SFV), infection compared with wild-type mice. Here, we evaluated SFV replication in different tissues of mice defective in both cytolytic pathways (perf(-/-)xgld) relative to that in wild-type counterparts and found that viral replication in perf(-/-)xgld mice is remarkably restricted. Although the mechanism responsible for this observation is yet to be established, the lower virus titres found in these mice indicate that the role of cytolytic effector molecules in antiviral immunity needs to be re-evaluated.

Interferon type I responses in primary and secondary infections.

The mammalian host responds to a microbial infection with a rapid innate immune reaction that is dominated by type I interferon (IFN-I) release. Most cells of vertebrates can respond to microbial attack with IFN-I production, but the cell type responsible for most of the systemic IFN-I release is thought to be plasmacytoid dendritic cells (pDCs). Besides its anti-microbial and especially anti-viral properties IFN-I also exerts a regulatory role on many facets of the sequential adaptive immune response. One of these is being the recently described partial, systemic activation of the vast majority of B and T lymphocytes in mice, irrespective of antigen reactivity. The biological significance of this partial activation of lymphocytes is at present speculative. Secondary infections occurring within a short time span of a primary infection fail to elicit a similar lymphocyte activation response due to a refractory period in systemic IFN-I production. This period of exhaustion in IFN-I responses is associated with an increased susceptibility of the host to secondary infections. The latter correlates with well-established clinical observations of heightened susceptibility of patients to secondary microbial infections after viral episodes.