Immunization with cell-free-generated vaccine protects from Porphyromonas gingivalis-induced alveolar bone loss.

INTRODUCTION: Periodontal diseases (PD) are complex oral inflammatory diseases initiated by keystone bacteria such as Porphyromonas gingivalis. A vaccine for PD is desirable as clinical treatment involves protracted maintenance strategies aimed to retain dentition. Although prior immunization approaches targeting P. gingivalis have reported variable success in limiting facets of disease such as oral bone loss, it remains that a vaccine for this disease may be attainable. AIM: To investigate cell-free protein synthesis (CFPS) as a platform to produce vaccinable targets suitable for efficacy testing in a P. gingivalis-induced murine oral bone loss model. MATERIALS AND METHODS: Recombinantly generated P. gingivalis minor fimbriae protein (Mfa1), RgpA gingipain hemagglutinin domain 1 (HA1), and RgpA gingipain hemagglutinin domain 2 (HA2) were combined in equivalent doses in adjuvants and injected intramuscularly to immunize mice. Serum levels of protein-specific antibody were measured by ELISA, and oral bone levels were defined by morphometrics. RESULTS: Recombinantly generated P. gingivalis proteins possessed high fidelity to predicted size and elicited protein-specific IgG following immunization. Importantly, immunization with the vaccine cocktail protected from P. gingivalis elicited oral bone loss. CONCLUSION: These data verify the utility of the CFPS technology to synthesize proteins that have the capacity to serve as novel vaccines.

A Lipid/DNA Adjuvant-Inactivated Influenza Virus Vaccine Protects Rhesus Macaques From Uncontrolled Virus Replication After Heterosubtypic Influenza A Virus Challenge.

Background: Influenza A virus (IAV) vaccines offer little protection from mismatched viruses with antigenically distant hemagglutinin (HA) glycoproteins. We sought to determine if a cationic lipid/DNA complex (CLDC) adjuvant could induce heterosubtypic protection if added to a whole inactivated IAV vaccine (WIV). Methods: Adult rhesus macaques (RMs) were vaccinated and at 2 weeks boosted with either an H1N1-WIV or an H3N2-WIV, with and without CLDC adjuvant. Four weeks postboost, animals were challenged with an H1N1 IAV matched to the H1N1-WIV vaccine. Results: After challenge, viral RNA (vRNA) levels in the trachea of control RMs and RMs vaccinated with the unadjuvanted H1 or H3 WIV vaccines were similar. However, vRNA levels in the trachea of both the H1-WIV/CLDC- and the H3-WIV/CLDC-vaccinated RMs (P < 0.01 and P < 0.05, respectively) were significantly lower than in unvaccinated control RMs. Heterosubtypic protection in H3-WIV/CLDC RMs was associated with significantly higher levels of nucleoprotein (NP) and matrix-1-specific immunoglobulin G antibodies (P < 0.05) and NP-specific nonneutralizing antibody-dependent natural killer cell activation (P < 0.01) compared with unprotected H3-WIV RMs. Conclusions: Addition of the CLDC adjuvant to a simple WIV elicited immunity to conserved virus structural proteins in RMs that correlate with protection from uncontrolled virus replication after heterosubtypic influenza virus challenge.

Prevention of liver tumor formation in woodchucks with established hepatocellular carcinoma by treatment with cationic liposome-DNA complexes.

BACKGROUND: Approximately 250 million people worldwide are chronically infected with hepatitis B virus (HBV) and more than half of the hepatocellular carcinoma (HCC) cases are attributed to this infection. As HCC has a high mortality rate, and current treatment options are remarkably limited, the development of new therapeutic treatment strategies is warranted. METHODS: In this study, woodchucks infected with woodchuck hepatitis virus (WHV), and with pre-existing liver tumors, were used as a model to investigate if complexes of cationic liposomes and non-coding DNA (JVRS-100) were effective in treatment of HCC. RESULTS: It was observed that the high serum viral load that is present in a typical chronic WHV infection (i.e., approximately 100-fold higher than human viral loads) results in immune suppression and resistance to treatment with JVRS-100. Treatment of woodchucks with lower serum viral load that more closely matched with the viral load usually seen in human HBV infection appears a better model for immunotherapeutic development based on the responsiveness to JVRS-100 treatment. In the latter case, marked declines in WHV DNA and WHV surface antigen were determined over the 12-week treatment period and WHV markers stayed suppressed during most time points of the 12-week follow-up period. Even more remarkably, the formation of new liver tumors was not observed in woodchucks treated with a well-tolerated dose of JVRS-100, as compared to several new tumors that developed in vehicle-treated control animals. CONCLUSIONS: Although there was little decrease in the volumes of the liver tumors existing at the time of treatment, it is generally accepted that preventing the spread and metastasis of almost always fatal cancers such as HCC and thus, reducing it to a chronic and treatable disease can also be a successful therapeutic approach. The results in woodchucks warrant the investigation of JVRS-100 as an intervention to prevent liver cancer in patients chronically infected with HBV and at high risk for HCC development.

A cationic liposome-DNA complexes adjuvant (JVRS-100) enhances the immunogenicity and cross-protective efficacy of pre-pandemic influenza A (H5N1) vaccine in ferrets.

Influenza A (H5N1) viruses continue to pose a public health threat. As inactivated H5N1 vaccines are poorly immunogenic, adjuvants are needed to improve the immunogenicity of H5N1 vaccine in humans. Here, we investigated the immunogenicity and cross-protective efficacy in ferrets of a clade 2.2-derived vaccine with addition of JVRS-100, an adjuvant consisting of cationic liposome-DNA complexes (CLDC). After the first vaccination, significantly higher levels of hemagglutination-inhibition (HAI) and neutralizing antibody titers were detected in ferrets immunized with adjuvanted vaccine compared to unadjuvanted vaccine. Following a second dose of adjuvanted vaccine, HAI antibody titers of ≥ 40 were detected against viruses from multiple H5N1 clades. HAI antibodies against newly isolated H5N2 and H5N8 viruses were also augmented by JVRS-100. Ferrets were challenged with a heterologous H5N1 virus. All ferrets that received two doses of adjuvanted vaccine exhibited mild illness, significantly reduced nasal wash virus titers and protection from lethal challenge. In contrast, ferrets that received unadjuvanted vaccine showed greater weight loss, high viral titers and 3 of 6 animals succumbed to the lethal challenge. Our results indicate that the addition of JVRS-100 to H5N1 vaccine enhanced immunogenicity and cross-protection against lethal H5N1 virus disease in ferrets. JVRS-100 warrants further investigation as a potential adjuvant for influenza vaccines.

Efficacy of influenza vaccination of elderly rhesus macaques is dramatically improved by addition of a cationic lipid/DNA adjuvant.

BACKGROUND: The decreased immune response among elderly individuals results in reduced influenza vaccine efficacy. Strategies to improve vaccine efficacy in elderly individuals are needed. The goal of this study was to determine whether a cationic lipid/DNA complex (CLDC) can improve the efficacy of the trivalent inactivated influenza vaccine Fluzone in elderly nonhuman primates. METHODS: Elderly (age, >18 years) rhesus macaques were vaccinated with Fluzone, with or without CLDC, and challenged with a human seasonal influenza virus isolate, A/Memphis/7/2001(H1N1). RESULTS: We found that elderly macaques have significantly lower levels of circulating naive CD4(+) T cells, naive CD8(+) T cells, and B cells as compared to juvenile monkeys. Furthermore, on the day of challenge, recipients of Fluzone/CLDC had significantly higher plasma anti-influenza virus immunoglobulin G (P < .001) and immunoglobulin A (P < .001) titers than recipients of Fluzone alone. After virus challenge, only the Fluzone/CLDC-vaccinated animals had a significantly lower level of virus replication (P < .01) relative to the unvaccinated control animals. CONCLUSIONS: These results demonstrate that CLDC can enhance the immunogenicity and efficacy of a licensed TIV in immunosenescent elderly monkeys.

Cationic liposome-DNA complexes (CLDC) adjuvant enhances the immunogenicity and cross-protective efficacy of a pre-pandemic influenza A H5N1 vaccine in mice.

The development of pre-pandemic influenza A H5N1 vaccines that confer both antigen-sparing and cross-clade protection are a high priority given the limited worldwide capacity for influenza vaccine production, and the antigenic and genetic heterogeneity of circulating H5N1 viruses. The inclusion of potent adjuvants in vaccine formulations may achieve both of these aims. Here we show that the addition of JVRS-100, an adjuvant consisting of cationic liposome-DNA complexes (CLDC) to a clade 1-derived H5N1 split vaccine induced significantly higher virus-specific antibody than unadjuvanted formulations, with a >30-fold dose-sparing effect and induction of increased antigen-specific CD4(+) T-cell responses in mice. All mice that received one dose of adjuvanted vaccine and subsequent H5N1 viral challenges exhibited mild illness, lower lung viral titers, undetectable spleen and brain viral titers, and 100% survival after either homologous clade 1 or heterologous clade 2 H5N1 viral challenges, whereas unadjuvanted vaccine recipients showed significantly increased weight loss, viral titers, and mortality. The protective immunity induced by JVRS-100 adjuvanted H5N1 vaccine was shown to last for over one year without significant waning. Thus, JVRS-100 adjuvanted H5N1 vaccine elicited enhanced humoral and T-cell responses, dose-sparing, and cross-clade protection in mice. CLDC holds promise as an adjuvant for human pre-pandemic inactivated H5N1 vaccines.

Breaking B and T cell tolerance using cationic lipid--DNA complexes (CLDC) as a vaccine adjuvant with hepatitis B virus (HBV) surface antigen in transgenic mice expressing HBV.

Cationic lipid DNA complexes (CLDC), referred to here as JVRS-100, were evaluated as an adjuvant for hepatitis B surface antigen (HBsAg) for eliciting B and T cell responses in transgenic mice expressing hepatitis B virus (HBV). To confirm the immunogenicity of HBsAg+JVRS-1000, a study was conducted in C57BL/6 mice, the genetic background of the HBV transgenic mice used in the study. HBsAg+JVRS-100 elicited a T cell response and B cell response as evidenced by interferon-gamma (IFN-γ) secretion by re-stimulated splenocytes and anti-HBsAg IgG induction, respectively, whereas, HBsAg only elicited a B cell response. In HBV transgenic mice, HBsAg did not elicit either T or B cell responses, unlike the HBsAg+JVRS-100 that elicited both. Energix-B vaccine did perform better than the HBsAg by eliciting a B cell response in the transgenic mice, but it did not perform as HBsAg+JVRS-100 since it did not elicit a T cell response. The response by HBsAg+JVRS-100 was not sufficient to cause destruction of infected liver cells, but it did suppress HBV DNA non-cytolytically. From these results, JVRS-100 might be considered for further development as an adjuvant for HBV therapeutic vaccines.

Cationic lipid/DNA complex-adjuvanted influenza A virus vaccination induces robust cross-protective immunity.

Influenza A virus is a negative-strand segmented RNA virus in which antigenically distinct viral subtypes are defined by the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins. An ideal inactivated vaccine for influenza A virus would induce not only highly robust strain-specific humoral and T-cell immune responses but also cross-protective immunity in which an immune response to antigens from a particular viral subtype (e.g., H3N2) would protect against other viral subtypes (e.g., H1N1). Cross-protective immunity would help limit outbreaks from newly emerging antigenically novel strains. Here, we show in mice that the addition of cationic lipid/noncoding DNA complexes (CLDC) as adjuvant to whole inactivated influenza A virus vaccine induces significantly more robust adaptive immune responses both in quantity and quality than aluminum hydroxide (alum), which is currently the most widely used adjuvant in clinical human vaccination. CLDC-adjuvanted vaccine induced higher total influenza virus-specific IgG, particularly for the IgG2a/c subclass. Higher levels of multicytokine-producing influenza virus-specific CD4 and CD8 T cells were induced by CLDC-adjuvanted vaccine than with alum-adjuvanted vaccine. Importantly, CLDC-adjuvanted vaccine provided significant cross-protection from either a sublethal or lethal influenza A viral challenge with a different subtype than that used for vaccination. This superior cross-protection afforded by the CLDC adjuvant required CD8 T-cell recognition of viral peptides presented by classical major histocompatibility complex class I proteins. Together, these results suggest that CLDC has particular promise for vaccine strategies in which T cells play an important role and may offer new opportunities for more effective control of human influenza epidemics and pandemics by inactivated influenza virus vaccine.

A non-coding cationic lipid DNA complex produces lasting anti-leukemic effects.

Cationic lipid DNA complex (CLDC) is an immunostimulatory preparation that has significant anti-leukemic effects in multiple murine models of leukemia: BCR-ABL(+) myelogenous leukemia in C3H/HeJ animals and myelomonocytic leukemia in BALB/c mice. Following leukemic challenge, CLDC treatment inhibits tumor cell growth in vivo and extends survival, sometimes resulting in apparent eradication of tumor cells. CLDC induces multiple cytokines including interferon-gamma (IFNγ), and intravenous treatment results in a more rapid and robust response than subcutaneous treatment. IFNγ is induced in a dose-dependent manner, and tachyphylaxis results from repeated doses of CLDC. Tachyphylaxis of therapeutic effects is exacerbated at higher doses, thus the optimal survival benefits are seen at intermediate doses. Animals whose leukemia has been successfully treated with CLDC exhibit a survival advantage when faced with a secondary leukemic challenge, suggesting the existence of an adaptive anti-leukemic response. This work demonstrates the effectiveness of CLDC in multiple experimental leukemias and is consistent with a stimulation of a lasting TH(1) anti-leukemic immune response.

Effective, broad spectrum control of virulent bacterial infections using cationic DNA liposome complexes combined with bacterial antigens.

Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens.

Rapid immunity to vaccination with woodchuck hepatitis virus surface antigen using cationic liposome-DNA complexes as adjuvant.

Complexes of cationic liposomes and non-coding DNA (CLDC) have shown promise as vaccine adjuvant. Using the woodchuck animal model of hepatitis B virus (HBV) infection, the immunogenic effects of CLDC were evaluated following vaccination with three doses of woodchuck hepatitis virus surface antigen (WHsAg) adjuvanted with either CLDC or conventional alum and administered intramuscularly (im) or subcutaneously (sc). IM vaccination with WHsAg and CLDC elicited antibodies earlier, in more woodchucks, and with higher titers than WHsAg and alum. After two vaccine doses, antibody titers were higher following im than sc administration. Woodchucks administered two vaccine doses sc received the third vaccine dose im, and antibody responses reached titers comparable to those elicited by im administration. Following the second vaccine dose, im vaccination with WHsAg and CLDC induced T cell responses to WHsAg and selected WHs peptides and expression of the leukocyte surface marker CD8 and of the Th1 cytokines interferon-gamma and tumor necrosis factor alpha in woodchucks. T cell responses and CD8/cytokine expression were diminished in woodchucks from the other groups suggesting that this vaccine regimen induced a skew toward Th1 immune responses. The present study in woodchucks demonstrates that CLDC-adjuvanted WHsAg vaccine administered im resulted in a more rapid induction of humoral and cellular immune responses compared to conventional, alum-adjuvanted WHsAg vaccine. While less rapid, the immune responses following sc administration can prime the im immune responses. This adjuvant activity of CLDC over alum may be beneficial for therapeutic vaccination in chronic HBV infection.

Cationic lipid/DNA complexes (JVRS-100) combined with influenza vaccine (Fluzone) increases antibody response, cellular immunity, and antigenically drifted protection.

Safe and effective adjuvants for influenza vaccines that could increase both the levels of neutralizing antibody, including against drifted viral subtypes, and T-cell immunity would be a major advance in vaccine design. The JVRS-100 adjuvant, consisting of DOTIM/cholesterol cationic liposome-DNA complexes, is particularly promising for vaccines that require induction of high levels of antibody and T-cell immunity, including CD8(+) cytotoxic T lymphocytes (CTL). Inclusion of protein antigens with JVRS-100 results in the induction of enhanced humoral and cell-mediated (i.e., CD4(+) and CD8(+) T cells) immune responses. The JVRS-100 adjuvant combined with a split trivalent influenza vaccine (Fluzone-sanofi pasteur) elicited increased antibody and T-cell responses in mice and non-human primates compared to vaccination with Fluzone alone. Mice vaccinated with JVRS-100-Fluzone and challenged with antigenically drifted strains of H1N1 (PR/8/34) and influenza B (B/Lee/40) viruses had higher grade protection, as measured by attenuation of weight loss and increased survival, compared to recipients of unadjuvanted vaccine. The results indicate that the JVRS-100 adjuvant substantially increases immunogenicity and protection from drifted-strain challenge using an existing influenza vaccine.

Efficacy of cationic lipid-DNA complexes (CLDC) on hepatitis B virus in transgenic mice.

Cationic lipid-DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5 microg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05 microg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models.