Neonatal immunization with a Sindbis virus-DNA measles vaccine induces adult-like neutralizing antibodies and cell-mediated immunity in the presence of maternal antibodies

Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. We evaluated the immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG2a, that also inhibited syncytium formation in CD150(+) B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong Th1 type cell-mediated immunity, measured by T cell proliferation and IFN-gamma production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life.     

Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.     

DNA vaccination strategies against infectious diseases

DNA immunisation represents a novel approach to vaccine and immunotherapeutic development. Injection of plasmid DNA encoding a foreign gene of interest can result in the subsequent expression of the foreign gene products and the induction of an immune response within a host. This is relevant to prophylactic and therapeutic vaccination strategies when the foreign gene represents a protective epitope from a pathogen. The recent demonstration by a number of laboratories that these immune responses evoke protective immunity against some infectious diseases and cancers provides support for the use of this approach. In this article, we attempt to present an informative and unbiased representation of the field of DNA immunisation. The focus is on studies that impart information on the development of vaccination strategies against a number of human and animal pathogens. Investigations that describe the mechanism(s) of protective immunity induced by DNA immunisation highlight the advantages and disadvantages of this approach to developing vaccines within a given system. A variety of systems in which DNA vaccination has resulted in the induction of protective immunity, as well as the correlates associated with these protective immune responses, will be described. Particular attention will focus on systems involving parasitic diseases. Finally, the potential of DNA immunisation is discussed as it relates to veterinary medicine and its role as a possible vaccine strategy against animal coccidioses.     

Plasmid DNA encoding IFN-gamma-inducible protein 10 redirects antigen-specific T cell polarization and suppresses experimental autoimmune encephalomyelitis

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that stimulates the directional migration of activated T cells, particularly Th1 cells. We demonstrate in this work that during activation this chemokine drives naive CD4(+) T cells into Th1 polarization. Administration of plasmid DNA encoding self IP-10 was found capable of breaking down immunological tolerance to IP-10, resulting in the generation of self-specific immunity to the gene product of the vaccine. Despite the CpG motif that drives T cells into Th1, the vaccine redirected the polarization of myelin basic protein-specific T cells into Th2 and conferred the vaccinated recipients a high state of resistance against experimental autoimmune encephalomyelitis, a T cell-mediated autoimmune disease of the CNS. The vaccine also suppressed full-blown ongoing disease in a mouse model of multiple sclerosis. Self-specific Ab to IP-10 developed in protected animals could inhibit leukocyte migration, alter the in vitro Th1/Th2 balance of autoimmune T cells, and adoptively transfer disease suppression. This demonstrates not only the pivotal role of a chemokine in T cell polarization and function but also its potential implications for plasmid DNA gene therapy.     

Genetic immunization of mice against Listeria monocytogenes using plasmid DNA encoding listeriolysin O.

The development of protective immunity against many intracellular bacterial pathogens commonly requires sublethal infection with viable forms of the bacteria. Such infection results in the in vivo activation of specific cell-mediated immune responses, and both CD4+ and CD8+ T lymphocytes may function in the induction of this protective immunity. In rodent models of experimental infection with Listeria monocytogenes, the expression of protective immunity can be mediated solely by the immune CD8+ T cell subset. One major target Ag of Listeria-immune CD8+ T cells is the secreted bacterial hemolysin, listeriolysin O (LLO). In an attempt to generate a subunit vaccine in this experimental disease model, eukaryotic plasmid DNA expression vectors containing genes encoding either the wild-type or modified forms of recombinant LLO were generated and used for genetic vaccination of naive mice. Results of these studies indicate that the intramuscular immunization of mice with specifically designed plasmid DNA constructs encoding recombinant forms of LLO stimulates peptide-specific CD8+ immune T cells that exhibit in vitro cytotoxic activity. More importantly, such immunization can provide protective immunity against a subsequent challenge with viable L. monocytogenes, demonstrating that this experimental approach may have direct application in prevention of acute disease caused by intracellular bacterial pathogens.     

Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun.

Protective immunity against Listeria monocytogenes strongly depends on CD8+ T lymphocytes, and both IFN-gamma secretion and target cell killing are considered relevant to protection. We analyzed whether we could induce a protective type 1 immune response by DNA vaccination with the gene gun using plasmids encoding for two immunodominant listerial Ags, listeriolysin and p60. To induce a Th1 response, we 1) coprecipitated a plasmid encoding for GM-CSF, 2) employed a prime/boost vaccination schedule with a 45-day interval, and 3) coinjected oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. DNA immunization of BALB/c mice with plasmids encoding for listeriolysin (pChly) and p60 (pCiap) efficiently induced MHC class I-restricted, Ag-specific CD8+ T cells that produced IFN-gamma. Coinjection of CpG-ODN significantly increased the frequency of specific IFN-gamma-secreting T cells. Although pChly induced specific CD8+ T cells expressing CTL activity, it failed to stimulate CD4+ T cells. Only pCiap induced significant CD4+ T cell and humoral responses, which were predominantly of Th2 type. Vaccination with either plasmid induced protective immunity against listerial challenge, and coinjection of CpG ODN improved vaccine efficacy in some situations. This study demonstrates the feasibility of gene gun administration of plasmid DNA for inducing immunity against an intracellular pathogen for which protection primarily depends on type 1 CD8+ T cells.     

Presence of CpG DNA and the local cytokine milieu determine the efficacy of suppressive DNA vaccination in experimental autoimmune encephalomyelitis.

We here study the adjuvant properties of immunostimulatory DNA sequences (ISS) and coinjected cytokine-coding cDNA in suppressive vaccination with DNA encoding an autoantigenic peptide, myelin basic protein peptide 68-85, against Lewis rat experimental autoimmune encephalomyelitis (EAE). EAE is an autoaggressive, T1-mediated disease of the CNS. ISS are unmethylated CpG motifs found in bacterial DNA, which can induce production of type 1 cytokines in vertebrates through the innate immune system. Because ISS in the plasmid backbone are necessary for efficient DNA vaccination, we studied the effect of one such ISS, the 5'-AACGTT-3' motif, in our system. Treatment with a DNA vaccine encoding myelin basic protein peptide 68-85 and containing three ISS of 5'-AACGTT-3' sequence suppressed clinical signs of EAE, while a corresponding DNA vaccine without such ISS had no effect. We further observed reduced proliferative T cell responses in rats treated with the ISS-containing DNA vaccine, compared with controls. We also studied the possible impact of coinjection of plasmid DNA encoding rat cytokines IL-4, IL-10, GM-CSF, and TNF-alpha with the ISS-containing DNA vaccine. Coinjection of IL-4-, IL-10-, or TNF-alpha-coding cDNA inhibited the suppressive effect of the DNA vaccine on EAE, whereas GM-CSF-coding cDNA had no effect. Coinjection of cytokine-coding cDNA with the ISS-deficient DNA vaccine failed to alter clinical signs of EAE. We conclude that the presence of ISS and induction of a local T1 cytokine milieu is decisive for specific protective DNA vaccination in EAE.     

Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity

The presence of both cell-mediated and humoral immunity is important in protection from and clearance of a number of infectious pathogens. We describe novel vaccine regimens using combinations of plasmid DNA, poxvirus and protein to induce strong Ag-specific T cell and Ab responses simultaneously in a murine model. Intramuscular (i.m.) immunization with plasmid DNA encoding the middle Ag of hepatitis B (DNA) concurrently with a commercial hepatitis B virus (HBV) vaccine (Engerix-B) followed by boosting immunizations with both modified vaccinia virus Ankara (MVA) encoding the middle Ag of HBV and Engerix-B induced high levels of CD4(+) and CD8(+) T cells and high titer Ab responses to hepatitis B surface Ag (HbsAg). Substitution of Engerix-B with adjuvant-free rHBsAg induced similar T cell responses and greatly enhanced Ab levels. Repeated immunizations with recombinant or nonrecombinant MVA mixed with Ag induced higher titers of Abs compared with immunization with either Ag or Engerix-B further demonstrating this novel adjuvant effect of MVA. The poxviruses NYVAC, fowlpox (FP9) and ALVAC, and to a lesser extent, adenovirus, also displayed similar adjuvant properties when used in combination with rHBsAg. The use of poxviruses as an adjuvant for protein to concurrently induce Ag-specific T cells and Abs could be applied to the development of vaccines for many diseases, including HIV and malaria, where both cell mediated and humoral immunity may be important for protection.     

Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws

Gene therapy is a promising new approach in the treatment of rheumatoid arthritis. Gene delivery to diseased joints offers the prospect of achieving high, local concentrations of a therapeutic gene product in a sustained manner, while minimizing exposure of nontarget organs. We report that a single administration of a modified adenovirus encoding the Epstein-Barr-derived homologue of IL-10 can suppress the development of disease for extended periods of time when injected locally within the periarticular tissue surrounding the ankle joints of mice with collagen type II-induced arthritis. Furthermore, we show that injection of an adenoviral vector carrying the IL-10 gene into a single paw can suppress development of arthritis in other, noninjected paws of the same individual. The systemic protection resulting from local gene therapy occurred in the absence of detectable levels of viral IL-10 in the serum. Circulating Ab levels to heterologous collagen were unaffected; however, treatment with viral IL-10 significantly suppressed the development of Abs to autologous mouse type II collagen. Thus, the treatment of a single joint by local delivery of the vIL-10 gene may protect multiple joints of the same individual while avoiding deleterious side effects often associated with systemic therapy.     

Suppression of immune response and protective immunity to a Japanese encephalitis virus DNA vaccine by coadministration of an IL-12-expressing plasmid

IL-12 plays a central role in both innate and acquired immunity and has been demonstrated to potentiate the protective immunity in several experimental vaccines. However, in this study, we show that IL-12 can be detrimental to the immune responses elicited by a plasmid DNA vaccine. Coadministration of the IL-12-expressing plasmid (pIL-12) significantly suppressed the protective immunity elicited by a plasmid DNA vaccine (pE) encoding the envelope protein of Japanese encephalitis virus. This suppressive effect was associated with marked reduction of specific T cell proliferation and Ab responses. A single dose of pIL-12 treatment with plasmid pE in initial priming resulted in significant immune suppression to subsequent pE booster immunization. The pIL-12-mediated immune suppression was dose dependent and evident only when the IL-12 gene was injected either before or coincident with the pE DNA vaccine. Finally, using IFN-gamma gene-disrupted mice, we showed that the suppressive activity of the IL-12 plasmid was dependent upon endogenous production of IFN-gamma. These results demonstrate that coexpression of the IL-12 gene can sometimes produce untoward effects to immune responses, and thus its application as a vaccine adjuvant should be carefully evaluated.     

Anti-tumor immunity against CT26 colon tumor in mice immunized with plasmid DNA encoding beta-galactosidase fused to an envelope protein of endogenous retrovirus

Endogenous retroviral gene products have been recognized as being expressed in human cancerous tissues. However, these products have not been shown to be antigenic targets for T-cells, possibly due to immune tolerance. Since carcinogen-induced colon tumor CT26 expresses an envelope protein, gp70, of an endogenous ecotropic murine leukemia virus that is comparable to human tumor-associated antigens, we examined whether a DNA vaccine containing the gp70 gene induces protective immunity against CT26 cells. Injection of mice with plasmid DNA (pDNA) encoding gp70 alone failed to induce anti-gp70 antibody (Ab) or anti-CT26 cytotoxic T lymphocyte (CTL) responses. However, immunization with pDNA encoding the beta-galactosidase (beta-gal)/gp70 fusion protein induced anti-gp70 Ab and anti-CT26 CTL responses and conferred protective immunity against CT26 cells. These results indicate that beta-gal acts as an immunogenic carrier protein that helps in the induction of immune responses against the poorly immunogenic gp70. Considering these results, it is possible that potential tolerance to the endogenous retroviral gene products expressed by human tumors may be overcome by DNA vaccines that contain an endogenous retroviral gene fused to genes encoding immunogenic carrier proteins.     

Comparison of immune responses against FMD by a DNA vaccine encoding the FMDV/O/IRN/2007 VP1 gene and the conventional inactivated vaccine in an animal model

Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals. The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP₁ gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein. Six mice groups were respectively immunized by the sub-cloned pcDNA3.1⁺-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together, conventional vaccine, PBS (as negative control), pcDNA3.1⁺ vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group). Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine (P<0.05). Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine, but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Th1 cellular immunity.     

Heterogeneous effects of IFN-gamma in adjuvant arthritis

In an attempt to evaluate the role of IFN-gamma in autoimmune arthritis, we tested the effects of IFN-gamma and anti-IFN-gamma mAb (DB-1) in various phases of arthritis development in a rat model for rheumatoid arthritis; the adjuvant arthritis (AA) model, induced by immunization with CFA. In addition, the effects of IFN-gamma were tested in vitro on T cell clones derived from rats afflicted with AA. T cell clone A2b, which has been shown to be arthritogenic secreted low amounts of IFN-gamma and its Ag-specific proliferation was inhibited by IFN-gamma. In contrast, clone A2c, which can inhibit the development of AA, produced high amounts of IFN-gamma and its proliferation was increased by IFN-gamma. In vivo administration of IFN-gamma 24 h before CFA caused an enhancement of arthritis, whereas giving IFN-gamma 24 to 48 h after CFA suppressed the disease. Administration of IFN-gamma between day +4 to +12 or between day +12 to +24 increased the severity of the first phase of the disease, but had no effect later. Administration of DB-1 1 to 2 days before adjuvant or between day +4 to +8 substantially decreased the disease, whereas DB-1 given from day +12 to +24 significantly enhanced it. Taken together, these results illustrate the heterogeneity of IFN-gamma in autoimmune arthritis and suggest a rational explanation for the possibly conflicting reports regarding the role(s) and effects of IFN-gamma in autoimmune processes. The multistage nature of T cell-mediated autoimmune arthritis may be due to the predominance of distinct T cell populations at different stages of the disease. The differences in the biologic activities of these T cells may be due to their patterns of lymphokine production.     

What happens to the DNA vaccine in fish? A review of current knowledge

The primary function of DNA vaccines, a bacterial plasmid DNA containing a construct for a given protective antigen, is to establish specific and long-lasting protective immunity against diseases where conventional vaccines fail to induce protection. It is acknowledged that less effort has been made to study the fate, in terms of cellular uptake, persistence and degradation, of DNA vaccines after in vivo administration. However, during the last year some papers have given new insights into the fate of DNA vaccines in fish. By comparing the newly acquired information in fish with similar knowledge from studies in mammals, similarities with regard to transport, blood clearance, cellular uptake and degradation of DNA vaccines have been found. But the amount of DNA vaccine redistributed from the administration site after intramuscular administration seems to differ between fish and mammals. This review presents up-to-date and in-depth knowledge concerning the fate of DNA vaccines with emphasis on tissue distribution, cellular uptake and uptake mechanism(s) before finally describing the intracellular hurdles that DNA vaccines need to overcome in order to produce their gene product.     

Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that is thought to manifest a proinflammatory role because it stimulates the directional migration of activated T cells, particularly Th1 cells. It is an open question whether this chemokine is also directly involved in T cell polarization. We show here that during the course of adjuvant-induced arthritis the immune system mounts a notable Ab titer against self-IP-10. Upon the administration of naked DNA encoding IP-10, this titer rapidly accelerates to provide protective immunity. Self-specific Ab to IP-10 developed in protected animals, as well as neutralizing Ab to IP-10 that we have generated in rabbits, could inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2 balance toward low IFN-gamma, low TNF-alpha, high IL-4-producing T cells, and adoptively transfer disease suppression. This not only demonstrates the pivotal role of this chemokine in T cell polarization during experimentally induced arthritis but also suggests a practical way to interfere in the regulation of disease to provide protective immunity. From the basic science perspective, this study challenges the paradigm of in vivo redundancy. After all, we did not neutralize the activity of other chemokines that bind CXCR3 (i.e., macrophage-induced gene and IFN-inducible T cell alpha chemoattractant) and yet significantly blocked not only adjuvant-induced arthritis but also the in vivo competence to mount delayed-type hypersensitivity.     

Subunit recombinant vaccine protects against monkeypox

The smallpox vaccine Dryvax, a live vaccinia virus (VACV), protects against smallpox and monkeypox, but is contraindicated in immunocompromised individuals. Because Abs to VACV mediate protection, a live virus vaccine could be substituted by a safe subunit protein-based vaccine able to induce a protective Ab response. We immunized rhesus macaques with plasmid DNA encoding the monkeypox orthologs of the VACV L1R, A27L, A33R, and B5R proteins by the intradermal and i.m. routes, either alone or in combination with the equivalent recombinant proteins produced in Escherichia coli. Animals that received only DNA failed to produce high titer Abs, developed innumerable skin lesions after challenge, and died in a manner similar to placebo controls. By contrast, the animals vaccinated with proteins developed moderate to severe disease (20-155 skin lesions) but survived. Importantly, those immunized with DNA and boosted with proteins had mild disease with 15 or fewer lesions that resolved within days. DNA/protein immunization elicited Th responses and binding Ab titers to all four proteins that correlated negatively with the total lesion number. The sera of the immunized macaques recognized a limited number of linear B cell epitopes that are highly conserved among orthopoxviruses. Their identification may guide future efforts to develop simpler, safer, and more effective vaccines for monkeypox and smallpox.     

Enhanced T-Cell Immunogenicity and Protective Efficacy of a Human Immunodeficiency Virus Type 1 Vaccine Regimen Consisting of Consecutive Priming with DNA and Boosting with Recombinant Fowlpox Virus

The induction of human immunodeficiency virus (HIV)-specific T-cell responses is widely seen as critical to the development of effective immunity to HIV type 1 (HIV-1). Plasmid DNA and recombinant fowlpox virus (rFPV) vaccines are among the most promising safe HIV-1 vaccine candidates. However, the immunity induced by either vaccine alone may be insufficient to provide durable protection against HIV-1 infection. We evaluated a consecutive immunization strategy involving priming with DNA and boosting with rFPV vaccines encoding common HIV-1 antigens. In mice, this approach induced greater HIV-1-specific immunity than either vector alone and protected mice from challenge with a recombinant vaccinia virus expressing HIV-1 antigens. In macaques, a dramatic boosting effect on DNA vaccine-primed HIV-1-specific helper and cytotoxic T-lymphocyte responses, but a decline in HIV-1 antibody titers, was observed following rFPV immunization. The vaccine regimen protected macaques from an intravenous HIV-1 challenge, with the resistance most likely mediated by T-cell responses. These studies suggest a safe strategy for the enhanced generation of T-cell-mediated protective immunity to HIV-1.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.