Incidence of second primary cancers in oral and pharyngeal cancer patients using a large medical claims database in Japan.

Background/purpose: As the occurrence of second primary cancers (SPCs) is strongly related to the survival rate of patients with oral and pharyngeal cancers, early detection and treatment are important. Therefore, this study aimed to clarify the incidence of SPCs and their risk factors in patients with oral and pharyngeal cancer. Materials and methods: This observational study was conducted using data from the administrative claims database of 21,736 participants with oral and pharyngeal cancer from January 2005 to December 2020. We evaluated the cumulative incidence of SPCs among patients with oral and pharyngeal cancers using the Kaplan-Meier method. The Cox proportional-hazard model was used for multivariate analysis. Results: Of the 1633 patients with oral and pharyngeal cancer who qualified for analysis, 388 developed SPCs (incidence rate, 7.994/1000 person-months). The multivariate analysis showed that the risk of developing SPCs was affected by age at diagnosis of oral and pharyngeal cancer, cancer treatment, and anatomical site of the primary cancer. Conclusion: Patients with oral and pharyngeal cancers are at a high risk of developing SPCs. The data from this study may be useful in providing accurate information to patients with oral and oropharyngeal cancer.

[A Case of Achieving CR through Chemoradiotherapy for Postoperative Oligo Lymph Node Metastasis in Gastric Cancer].

We present a case of oligo lymph node metastasis in a 70s man who had previously undergone subtotal gastrectomy for advanced gastric cancer in the prepylorus. Postoperatively, adjuvant chemotherapy was administered for a duration of 1 year. During the third postoperative year, elevated tumor markers and lymph node enlargement prompted a diagnosis of lymph node metastasis. Subsequent chemoradiotherapy resulted in a complete response(CR), which has been sustained for 2 years without any recurrence. The outcomes of this case indicate that chemoradiotherapy stands as a viable treatment option for oligo lymphatic recurrence in gastric cancer.

Association between smoking habits and dental care utilization and cost using administrative claims database and specific medical check-up data.

BACKGROUND: This study aims to evaluate the association between smoking habits and dental care utilization and cost in individuals registered with the Japan Health Insurance Association, Osaka branch. METHODS: We used the administrative claims database and specific medical check-up data and included 226,359 participants, who visited dental institutions, underwent dental examinations, and underwent specific medical checkups, with smoking data from April 2016 to March 2017. We calculated propensity scores with age, gender, exercise, eating habits, alcohol intake, and sleep. We also compared dental care utilization with the total cost of each procedure. RESULTS: According to propensity score matching, 62,692 participants were selected for each group. Compared to non-smokers, smokers were younger, and a higher proportion were men. Smokers tended to skip breakfast, have dinner just before bed, and drink alcohol. After adjusting for potential confounding factors with propensity score matching, the mean annual dental cost among smokers was significantly higher than non-smokers. The prevalence of pulpitis, missing teeth, and apical periodontitis were higher among smokers than non-smokers, while inlay detachment, caries, and dentine hypersensitivity were higher among non-smokers. CONCLUSION: This study suggests that smokers have higher dental cost consisted of progressive dental caries, missing teeth, and uncontrolled acute inflammation that necessitated the use of medications. It is suggested that smokers tend to visit the dentist after their symptoms become severe.

Identification of HLA-A*24:02-Restricted CTL Candidate Epitopes Derived from the Nonstructural Polyprotein 1a of SARS-CoV-2 and Analysis of Their Conservation Using the Mutation Database of SARS-CoV-2 Variants.

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

Identification of HLA-A*02:01-restricted candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 that may be natural targets of CD8+ T cell recognition in vivo.

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three response patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.IMPORTANCE For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three reaction patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

Induction of adaptive immune responses against antigens incorporated within the capsid of simian virus 40.

Simian virus 40 (SV40) is a monkey polyomavirus. The capsid structure is icosahedral and comprises VP1 units that measure 45 nm in diameter. Five SV40 VP1 molecules form one pentamer subunit, and a single icosahedral subunit comprises 72 pentamers; a single SV40 VP1 capsid comprises 360 SV40 VP1 molecules. In a previous study, we showed that an influenza A virus matrix protein 1 (M1) CTL epitope inserted within SV40 virus-like particles (VLPs) induced cytotoxic T lymphocytes (CTLs) without the need for an adjuvant. Here, to address whether SV40 VLPs induce adaptive immune responses against VLP-incorporated antigens, we prepared SV40 VLPs containing M1 or chicken ovalbumin (OVA). This was done by fusing M1 or OVA with the carboxyl terminus of SV40 VP2 and co-expressing them with SV40 VP1 in insect cells using a baculovirus vector. Intraperitoneal (i.p.) or intranasal administration of SV40 VLPs incorporating M1 induced the production of CTLs specific for the M1 epitope without the requirement for adjuvant. The production of antibodies against SV40 VLPs was also induced by i.p. administration of SV40 VLPs in the absence of adjuvant. Finally, the administration of SV40 VLPs incorporating OVA induced anti-OVA antibodies in the absence of adjuvant; in addition, the level of antibody production was comparable with that after i.p. administration of OVA plus alum adjuvant. These results suggest that the SV40 capsid incorporating foreign antigens can be used as a vaccine platform to induce adaptive immune responses without the need for adjuvant.

Tannic acid acts as an agonist of the dopamine D2L receptor, regulates immune responses, and ameliorates experimentally induced colitis in mice.

Tannic acid (TA) is an herbal polyphenol containing a galloyl group that has been prescribed to treat gastroenteritis, diarrhea, and irritable bowel syndrome. TA has anti-inflammatory, anti-cancer, and anti-viral properties; however, the molecular mechanisms of these potential therapeutic effects are still largely unknown. Here, we examined the ability of TA to induce anti-inflammatory responses. TA was found to be an agonist of the dopamine D2L receptor. TA reduced interferon (IFN)-γ and interleukin (IL)-1ß secretion but upregulated tumor necrosis factor α and IL-10 secretion from lipopolysaccharide (LPS)-stimulated mouse splenocytes. TA also reduced IFN-γ secretion but enhanced IL-10 secretion from anti-cluster of differentiation (CD) 3/CD28 antibody-stimulated splenocytes. An immune subset study confirmed that TA regulated cytokine secretion by various types of immune cells in the context of stimulation with LPS or anti-CD3/CD28 antibodies. Administration of TA to mice with experimentally induced colitis strikingly suppressed weight loss, colon shrinkage, and IL-17 secretion from mesenteric lymph node lymphocytes in response to CD3/CD28 stimulation. These data suggest that TA suppresses inflammatory responses in colitis by regulating cytokine secretion by immune cells in the colon.

Dopamine regulates cytokine secretion during innate and adaptive immune responses.

Dopamine (DA) is synthesized by various immune cells. DA receptors (DARs), which comprise five isoforms, are expressed on the surface of these cells. Therefore, it is likely that DA plays a role in regulating innate and adaptive responses. However, the underlying molecular mechanism(s) is largely unknown. Here, we found that, during innate immune responses, DA suppressed secretion of IFN-γ, TNF-α and IL-1ß, but promoted secretion of IL-10 and CXCL1 by lipopolysaccharide (LPS)-stimulated mouse splenocytes, suggesting that DA regulates cytokine secretion. Immune subset studies indicated that DA suppressed secretion of IFN-γ, TNF-α and IL-1ß by NK cells, as well as secretion of TNF-α by neutrophils and monocytes; however, DA up-regulated IL-10 secretion by neutrophils, monocytes, B cells, macrophages (Mφs) and dendritic cells within the splenocyte population. In addition, DA up-regulated secretion of CXCL1 by LPS-stimulated NK cells and Mφs. Meanwhile, treatment with DAR agonists or antagonists suppressed secretion of inflammatory cytokines from LPS-stimulated splenocytes. Pre-treatment of LPS-stimulated splenocytes with the PI3K inhibitor wortmannin reversed DA-mediated suppression of IFN-γ secretion, indicating that DA regulates IFN-γ secretion via the inositol 1,4,5-trisphosphate signaling pathway in these cells. Administration of DA and LPS to mice immunized with chicken ovalbumin (OVA) increased secretion of IL-5 by mouse lung lymphocytes, suggesting that DA promotes OVA-specific Th2-mediated immune responses by these cells. Taken together, these findings indicate that DA regulates cytokine secretion during innate and adaptive immune responses.

Characterization of the flow cytometric assay for ex vivo monitoring of cytotoxicity mediated by antigen-specific cytotoxic T lymphocytes.

Several non-radioactive methods have widely been utilized to detect antigen-specific cytotoxic T lymphocyte (CTL) responses instead of the classical 51Cr-release assay. These methods include intracellular cytokine staining, major histocompatibility complex-class I tetramers, and the CD107a mobilization assay. However, they do not directly measure target-cell death. In contrast, several attempts have been made to develop the flow cytometric CTL (FC-CTL) assay for evaluation of cytotoxicity. However, further improvement is necessary for it to become standardized. Here, we evaluated the characteristics of the FC-CTL assay based on the uptake of propidium iodide (PI) using target cell lines expressing the green fluorescent protein (GFP). The FC-CTL assay was found to be sensitive enough to detect primary CTL responses. The usage of a pre-established GFP-expressing target cell line facilitated the procedure of the assay, and enabled a clear discrimination between target and effector cells. Time-course analyses demonstrated that PI-stained target cells were detected as early as surface CD107a expression after antigenic stimulation. Thus, the PI/GFP-based FC-CTL assay is sufficiently sensitive to practically detect the early stages of target-cell death, and may have a great potential for becoming a standard tool to measure CTL activity.

SV40 VP1 major capsid protein in its self-assembled form allows VP1 pentamers to coat various types of artificial beads in vitro regardless of their sizes and shapes.

The icosahedral capsid structure of simian virus 40 (diameter, 45 nm) consists of 72 pentameric subunits, with each subunit formed by five VP1 molecules. Electron microscopy, immuno-gold labeling, and ζ-potential analysis showed that purified recombinant VP1 pentamers covered polystyrene beads measuring 100, 200, and 500 nm in diameter, as well as silica beads. In addition to covering spherical beads, VP1 pentamers covered cubic magnetite beads, as well as the distorted surface structures of liposomes. These findings indicate that VP1 pentamers could coat artificial beads of various shapes and sizes larger than the natural capsid. Technology based on VP1 pentamers may be useful in providing a capsid-like surface for enclosed materials, enhancing their stability and cellular uptake for drug delivery systems.

Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants.

Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties.

Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity.

We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L) that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, ß2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP)-derived peptide, ß2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

Viral protein-coating of magnetic nanoparticles using simian virus 40 VP1.

Artificial beads including magnetite and fluorescence particles are useful to visualize pathologic tissue, such as cancers, from harmless types by magnetic resonance imaging (MRI) or fluorescence imaging. Desirable properties of diagnostic materials include high dispersion in body fluids, and the ability to target specific tissues. Here we report on the development of novel magnetic nanoparticles (MNPs) intended for use as diagnosis and therapy that are coated with viral capsid protein VP1-pentamers of simian virus 40, which are monodispersive in body fluid by conjugating epidermal growth factor (EGF) to VP1. Critically, the coating of MNPs with VP1 facilitated stable dispersion of the MNPs in body fluids. In addition, EGF was conjugated to VP1 coating on MNPs (VP1-MNPs). EGF-conjugated VP1-MNPs were successfully used to target EGF receptor-expressing tumor cells in vitro. Thus, using viral capsid protein VP1 as a coating material would be useful for medical diagnosis and therapy.

SV40 virus-like particles as an effective delivery system and its application to a vaccine carrier.

The authors have purified a major capsid protein, VP1 of Simian virus 40 (SV40), using recombinant baculovirus and have established the method of in vitro reassembly of SV40 virus-like particles (SV40-VLPs) from VP1-pentamers. In this reassembly, SV40-VLPs can encapsulate approximately 5 kb exogenous DNA shielded by histone or foreign proteins fused to minor capsid proteins VP2/3 and effectively deliver them into mammalian cells. Insertion of a particular foreign peptide into the surface loops of VP1 provides SV40-VLPs with the ability of cell targeting. Furthermore, SV40-VLPs appear to stimulate innate immunity as a natural adjuvant. Given these characteristics, SV40-VLPs may be a promising vaccine carrier to deliver heterologous antigens for the induction of cytotoxic T lymphocytes without artificial adjuvants. In this review, the authors describe how SV40-VLPs have been developed and engineered, and discuss their potential benefits and challenges as a cytotoxic T lymphocyte-based vaccine platform.

The route of immunization with adenoviral vaccine influences the recruitment of cytotoxic T lymphocytes in the lung that provide potent protection from influenza A virus.

Virus-specific cytotoxic T lymphocytes (CTLs) in the lung are considered to confer protection from respiratory viruses. Several groups demonstrated that the route of priming was likely to have an implication for the trafficking of antigen-specific CTLs. Therefore, we investigated whether the route of immunization with adenoviral vaccine influenced the recruitment of virus-specific CTLs in the lung that should provide potent protection from influenza A virus. Mice were immunized with recombinant adenovirus expressing the matrix (M1) protein of influenza A virus via various immunization routes involving intraperitoneal, intranasal, intramuscular, or intravenous administration as well as subcutaneous administration in the hind hock. We found that the immunization route dramatically impacted the recruitment of M1-specific IFN-γ(+) CD8(+) T cells both in the lung and the spleen. Surprisingly, hock immunization was most effective for the accumulation in the lung of IFN-γ-producing CD8(+) T cells that possessed M1-specific cytolytic activity. Further, antigen-driven IFN-γ(+) CD8(+) T cells in the lung, but not in the spleen, were likely to be correlated with the resistance to challenge with influenza A virus. These results may improve our ability to design vaccines that target virus-specific CTL responses to respiratory viruses such as influenza A virus.

Free immunoglobulin light chain: its biology and implications in diseases.

Immunoglobulin light chain (IgLC) is a component of antibodies, but its free form is observed in the circulation, which originates from 10 to 40% excess synthesis over heavy chain in B cells. Complete antibodies function as a defined tetramer structure unit, H2L2; thus, separation of heavy and light chains results in considerable or complete loss of antigen-binding ability. Free IgLC has been considered as an inconsequential spillover during antibody assembly because, unlike heavy chain, neither effector functions such as complement activation nor specific-receptor binding has been identified in IgLCs. Free IgLC in sera and cerebrospinal fluids increases in inflammatory diseases such as autoimmune diseases and infections, presumably as a result of B-cell activation. This may be just a concomitant event during elevated disease activity, but recent findings suggest that free IgLC is involved in a wide range of immunological phenomena as a signaling effector or an anti-inflammatory molecule. These effects are likely to be intrinsic to IgLC. In this review, we attempt to give a comprehensive view about the biological roles of free IgLC together with the gene expression, secretion, antigen-binding ability, and its metabolic characteristics.

Local expression of interleukin-27 ameliorates collagen-induced arthritis.

OBJECTIVE: To determine the mechanism of action of interleukin-27 (IL-27) against rheumatoid arthritis (RA). METHODS: Adenovirus containing IL-27 transcript was constructed and was locally delivered into the ankles of mice with collagen-induced arthritis (CIA). Progression of arthritis was determined in treated and untreated mice by measuring ankle circumference and through histologic analysis. IL-17 and its downstream targets as well as cytokines promoting Th17 cell differentiation were quantified by enzyme-linked immunosorbent assay in CIA mouse ankles locally expressing adenoviral IL-27 as well as in control-treated mouse ankles. Ankles from both treatment groups were immunostained for neutrophil and monocyte migration (macrophages in the tissue). Finally, vascularization was quantified by histology and by determining ankle hemoglobin levels. RESULTS: Ectopic expression of IL-27 in CIA mice ameliorated inflammation, lining hypertrophy, and bone erosion as compared with control-treated CIA mice. Serum and joint levels of IL-17 were significantly reduced in the IL-27-treated group compared with the control-treated group. Two of the main cytokines that induce Th17 cell differentiation and IL-17 downstream target molecules were greatly down-regulated in CIA mouse ankles receiving forced expression of IL-27. The control mice had higher levels of vascularization and monocyte trafficking than did mice ectopically expressing IL-27. CONCLUSION: Our results suggest that increased levels of IL-27 relieve arthritis in CIA mouse ankles. This amelioration of arthritis involves a reduction in CIA mouse serum and joint levels of IL-17 and results in decreased IL-17-mediated monocyte recruitment and angiogenesis. Hence, the use of IL-27 may be a strategy for treatment of patients with RA.

A pivotal role for interleukin-27 in CD8+ T cell functions and generation of cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) play a critical role in the control of various cancers and infections, and therefore the molecular mechanisms of CTL generation are a critical issue in designing antitumor immunotherapy and vaccines which augment the development of functional and long-lasting memory CTLs. Interleukin (IL)-27, a member of the IL-6/IL-12 heterodimeric cytokine family, acts on naive CD4+ T cells and plays pivotal roles as a proinflammatory cytokine to promote the early initiation of type-1 helper differentiation and also as an antiinflammatory cytokine to limit the T cell hyperactivity and production of pro-inflammatory cytokines. Recent studies revealed that IL-27 plays an important role in CD8+ T cells as well. Therefore, this article reviews current understanding of the role of IL-27 in CD8+ T cell functions and generation of CTLs.

A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice.

The current vaccination strategy against influenza is to induce the production of antibodies directed against surface antigens of viruses. However, the frequent changes in the surface antigens of influenza viruses allow the viruses to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. In the present study, liposomal conjugates with CTL epitope peptides derived from highly conserved internal antigens of influenza viruses were evaluated for their ability to protect against infection with influenza viruses. Liposomal conjugates with peptide M1 58-66, an HLA-A*0201-binding CTL epitope present within the amino-acid sequence of the M1 coding region, successfully induced antigen-specific CD8(+) T-cells and CTLs in HLA-A*0201-transgenic mice. Moreover, after nasal infection with either the H1N1 or H3N2 virus, viral replication in the lung was significantly inhibited in the immunized mice. These protective activities lasted at least 6months after the immunization. Thus, these results suggest that liposome-coupled CTL epitope peptides derived from highly conserved internal antigens of influenza viruses might be applicable to the development of vaccines that induce protection against infection with heterosubtypic influenza viruses.

Efficient induction of cytotoxic T lymphocytes specific for severe acute respiratory syndrome (SARS)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a.

Spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and major targets for cytotoxic T lymphocytes (CTLs). In contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. We previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited SARS-CoV-specific CTLs in mice. Here, we attempted to identify HLA-A*0201-restricted CTL epitopes derived from a non-structural polyprotein 1a (pp1a) of SARS-CoV, and investigated whether liposomal peptides derived from pp1a were effective for CTL induction. Out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (IFN-gamma)-producing CD8(+) T cells in mice. These peptides were coupled to the surface of liposomes, and inoculated into mice. Six liposomal peptides effectively induced IFN-gamma-producing CD8(+) T cells and seven liposomal peptides including the six peptides primed CTLs showing in vivo killing activities. Further, CTLs induced by the seven liposomal peptides lysed an HLA-A*0201 positive cell line expressing naturally processed, pp1a-derived peptides. Of note, one of the liposomal peptides induced high numbers of long-lasting memory CTLs. These data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient CTL-based vaccine against SARS.