Combined immunization with adjuvant molecules poly(I:C) and anti-CD40 plus a tumor antigen has potent prophylactic and therapeutic antitumor effects

The low immunogenicity of malignant cells is one of the causes responsible for the lack of antitumor immune responses. Thus, development of new therapeutic strategies aimed at enhancing presentation of tumor antigens to T cells is a main goal of cancer immunotherapy. With this aim, we studied the efficacy of administering adjuvants poly(I:C) and agonistic anti-CD40 antibody plus a tumor antigen. Joint intravenous immunization with these adjuvants and a model tumor antigen (ovalbumin) was able to synergistically induce potent and long lasting antitumor T-cell responses. These responses protected against challenge with E.G7–OVA tumor cells in prophylactic short- and long-term vaccination. In a therapeutic setting, repeated intratumor administration of adjuvants plus antigen was able to reject established tumors in all treated animals, leading in some cases to the rejection of both locally treated and untreated tumors. Antitumor immune responses induced by these protocols were mediated not only by T-cells but also by NK cells. In conclusion, combined administration of adjuvants poly(I:C) and anti-CD40 plus a tumor antigen is an efficient strategy for prophylactic and therapeutic antitumor vaccination.     

Experimental Salmonellosis X. Cellular Immunity and Its Antibody in Mouse Mononuclear Phagocytes

A cell-associated antibody was detected in the peritoneal mononuclear phagocytes (referred to as monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis, by use of immune transfer and immune adherence hemagglutination techniques. The cellular antibody inhibited the growth of a virulent strain of S. enteritidis with the aid of complement and lysozyme on nutrient agar plates. This type of bactericidal antibody could not be detected in the monocytes of mice immunized with killed vaccine of S. enteritidis. The antibody extracted from the peritoneal monocytes of mice hyperimmunized with live vaccine was identified as a macroglobulin by ultracentrifugal analysis.     

Delivering HIV Gagp24 to DCIR Induces Strong Antibody Responses In Vivo

Targeting dendritic cell-specific endocytic receptors using monoclonal antibodies fused to desired antigens is an approach widely used in vaccine development to enhance the poor immunogenicity of protein-based vaccines and to induce immune responses. Here, we engineered an anti-human DCIR recombinant antibody, which cross-reacts with the homologous cynomolgous macaque receptor and was fused via the heavy chain C-terminus to HIV Gagp24 protein (αDCIR.Gagp24). In vitro, αDCIR.Gagp24 expanded multifunctional antigen-specific memory CD4⁺ T cells recognizing multiple Gagp24 peptides from HIV-infected patient peripheral blood mononuclear cells. In non human primates, priming with αDCIR.Gagp24 without adjuvant elicited a strong anti-Gagp24 antibody response after the second immunization, while in the non-targeted HIV Gagp24 protein control groups the titers were weak. The presence of the double-stranded RNA poly(I:C) adjuvant significantly enhanced the anti-Gagp24 antibody response in all the groups and reduced the discrimination between the different vaccine groups. The avidity of the anti-Gagp24 antibody responses was similar with either αDCIR.Gagp24 or Gagp24 immunization, but increased from medium to high avidity in both groups when poly(I:C) was co-administered. This data provides a comparative analysis of DC-targeted and non-targeted proteins for their capacity to induce antigen-specific antibody responses in vivo. This study supports the further development of DCIR-based DC-targeting vaccines for protective durable antibody induction, especially in the absence of adjuvant.     

Nasal immunization with the mixture of PA63, LF, and a PGA conjugate induced strong antibody responses against all three antigens

A new generation anthrax vaccine is expected to target not only the anthrax protective antigen (PA) protein, but also other virulent factors of Bacillus anthracis. It is also expected to be amenable for rapid mass immunization of a large number of people. This study aimed to address these needs by designing a prototypic triantigen nasal anthrax vaccine candidate that contained a truncated PA (rPA63), the anthrax lethal factor (LF), and the capsular poly-gamma-D-glutamic acid (gammaDPGA) as the antigens and a synthetic double-stranded RNA (dsRNA), polyriboinosinic-polyribocytodylic acid (poly(I:C)) as the adjuvant. This study identified the optimal dose of nasal poly(I:C) in mice, demonstrated that nasal immunization of mice with the LF was capable of inducing functional anti-LF antibodies (Abs), and showed that nasal immunization of mice with the prototypic triantigen vaccine candidate induced strong immune responses against all three antigens. The immune responses protected macrophages against an anthrax lethal toxin challenge in vitro and enabled the immunized mice to survive a lethal dose of anthrax lethal toxin challenge in vivo. The anti-PGA Abs were shown to have complement-mediated bacteriolytic activity. After further optimization, this triantigen nasal vaccine candidate is expected to become one of the newer generation anthrax vaccines.     

Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.     

Augmentation of mouse natural killer cell activity by interferon and interferon inducers.

Interferon (IF), in addition to its anti-viral capacity, is increasingly being found to be a regulator of cell division, cell surface antigens, and cell function. To determine whether IF also plays a role in the regulation of natural killer (NK) cell activity in mice, the in vivo and in vitro effects of IF and IF inducers on NK activity were studied. We observed that pyran, lipopolysaccharide, and polyinosinicopolycytidylic acid (poly I:C) as well as crude and purified IF preparations significantly elevated splenic NK levels in normal mice within 3 to 24 hr of i.p. administration. Normal spleen cells treated with poly I:C or IF in vitro also had augmented NK activity. Poly I:C and IF were themselves not cytotoxic and their presence was not required during the lytic process, indicating that IF acts on lymphocytes to activate NK function. The addition of anti-IF in the incubation medium completely blocked the boosting of NK activity by poly I:C or IF. The characteristics of the effector cells activated by IF were consistent with those of NK cells rather than macrophages, since the boosted effector cells were not retained by a rayon column or removed by carbonyl iron. Moreover, they were resistant to treatment with anti-Thy 1.2 serum plus complement, which eliminated mature T cells.     

Relationship between tumour resistance and the in vitro and in vivo cytotoxicity elicited by Salmonella antigens in immunized mice

Immunization of mice with the attenuated 11RX strain of Salmonella enteritidis (11RX) induces resistance to intraperitoneal (i.p.) tumour growth. Tumour resistance is much greater and lasts for a longer time following i.p. immunization than following intravenous (i.v.) immunization. This paper extends our previous observations that, after this resistance is lost, it can be recalled by a T cell-mediated reaction to an antigenic extract of the bacteria (11RX antigen) which is not protective in unimmunized mice. The duration of this sensitization to 11RX antigen was determined in mice immunized i.p. or i.v. with live 11RX by challenging them at various times after immunization with 131I (or 125I)-labelled Ehrlich ascites tumour (EAT) cells alone or mixed with 11RX antigen. In vivo killing of EAT cells was assayed by monitoring whole-body retention of radioactivity and this was correlated in the same mice with suppression of tumour growth and survival of the mice. The resistance recalled by 11RX antigen was short-lived and in vivo cytotoxic activity had subsided by 6 days after antigen injection. 11RX antigen also recalled the ability of the peritoneal cells to lyse 51Cr-labelled EAT cells in vitro and a close correlation was found between this activity and the cytotoxicity measured in vivo. The adherence properties of the cytotoxic cells and their inhibition by trypan blue indicated that they were macrophages.     

Intradermal antitetanic-antityphoid booster by jet injection.

The immunogenicity and the reactogenicity of an unadsorbed Tetanus vaccine, intradermally administered in man as a booster immunization associated or not with lyophilized Typhoid vaccine, were comparatively studied. There were no differences in postvaccinal reactions between the Tetanus vaccine administered alone or associated with Typhoid vaccine as well as between the unadsorbed and adsorbed Tetanus vaccine. The booster inoculation carried out with Tetanus vaccine by i.d. route with doses of 10 Lf/0.1 ml proved to be effective, inducing to all the subjects a definite protective titre, maintaining for at least one year.     

Intranasal administration of a detoxified endotoxin vaccine protects mice against heterologous Gram-negative bacterial pneumonia

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli J5 mutant lipopolysaccharide (J5dLPS)-group B meningococcal outer membrane protein (-OMP) vaccine previously protected animals from lethal sepsis. To assess the use of this vaccine for the treatment of Gram-negative bacillary pneumonia, we vaccinated mice, with or without the adjuvant CpG, by intranasal (i.n.) or intraperitoneal (i.p.) routes of administration. Local and systemic IgG levels were 2-3 logs higher following i.p. immunization compared to i.n. However, i.n. immunization elicited both local and systemic IgA, unlike i.p. administration. The addition of CpG to the vaccine, by either route of administration, elicited greater levels of antibody. Intranasal immunization protected mice against lethal heterologous Gram-negative bacillary pneumonia and post-immunization serum and broncho-alveolar lavage fluid mediated enhanced bacterial killing with peritoneal and alveolar macrophages in vitro. We conclude that further studies on the use of J5dLPS-OMP for the prevention of nosocomial pneumonia are warranted.     

Heat-labile antigens of Salmonella enteritidis. I. Extraction of antigens

Milne, Margaret (University of Adelaide, Adelaide, Australia), and F. M. Collins. Heat-labile antigens of Salmonella enteritidis. I. Extraction of antigens. J. Bacteriol. 92:543-548. 1966.-Salmonella enteritidis strains of high and low mouse virulence were grown in continuous culture. Cell walls were obtained from both strains by sonic disruption, and the washed walls were extracted at 4 C with sodium dodecyl sulfate or sodium deoxycholate. The extracts were eluted with a saline gradient from a diethylaminoethyl cellulose column, and the separated peaks were tested for precipitin activity against rabbit antisera prepared with heator ethyl alcohol-killed vaccines. The separated antigens reacted less intensely with the antiserum to heat-killed cells than they did to the antisera prepared against alcohol-killed vaccine. Little antigenic difference could be detected between the extracts prepared from the virulent and the avirulent organisms.     

Identification of Salmonella typhimurium genes responsible for interference with peptide presentation on MHC class I molecules: Deltayej Salmonella mutants induce superior CD8+ T-cell responses

Salmonella-derived epitopes are presented on MHC molecules by antigen-presenting cells, and both CD4+ and CD8+ T cells participate in protective immunity to Salmonella. Therefore, mechanisms that allow Salmonella to escape specific immune recognition are likely to have evolved in this bacterial pathogen. To identify Salmonella genes, which potentially interfere with the MHC class I (MHC-I) presentation pathway, Tn10d transposon mutagenesis was performed. More than 3000 mutants, statistically covering half of the Salmonella genome, were individually screened for altered peptide presentation by infected macrophages. Two mutants undergoing enhanced antigen presentation by macrophages were identified, carrying a Tn10d insertion in the yej operon. This phenotype was validated by specific inactivation and complementation experiments. In accordance with their enhanced MHC-I presentation phenotype, we showed that (i) specific CD8+ T cells were elicited at a higher level in mice, in response to immunization with yej mutants compared to their parental strain in two different experimental settings; and (ii) yej mutants were superior vaccine carriers for heterologous antigens compared to the parental strain in a tumour model.     

Mucosal HIV-1 pox virus prime-boost immunization induces high-avidity CD8+ T cells with regime-dependent cytokine/granzyme B profiles

The quality of virus-specific CD8(+) CTL immune responses generated by mucosal and systemic poxvirus prime-boost vaccines were evaluated in terms of T cell avidity and single-cell analysis of effector gene expression. Intranasal (I.N.) immunization regimes generated higher avidity CTL responses specific for HIV K(d)Gag(197-205) (amino acid sequence AMQMLKETI; H-2K(d) binding) compared with i.m. immunization regime. Single-cell RT-PCR of K(d)Gag(197-205)-specific mucosal and systemic CTL revealed that the cytokine and granzyme B expression profiles were dependent on both the route and time after immunization. The I.N./i.m.-immunized group elicited elevated number of CTL-expressing granzyme B mRNA from the genitomucosal sites compared with the i.m./i.m. regime. Interestingly, CTL generated after both I.N. or i.m. immunization demonstrated expression of Th2 cytokine IL-4 mRNA that was constitutively expressed over time, although lower numbers were observed after I.N./I.N. immunization. Results suggest that after immunization, Ag-specific CTL expression of IL-4 may be an inherent property of the highly evolved poxvirus vectors. Current observations indicate that the quality of CTL immunity generated after immunization can be influenced by the inherent property of vaccine vectors and route of vaccine delivery. A greater understanding of these factors will be crucial for the development of effective vaccines in the future.     

Macrophage Ia antigens. I. macrophage populations differ in their expression of Ia antigens

By indirect immunofluorescence and microcytotoxicity it was demonstrated that different populations of murine macrophages bear different amounts of Ia antigens on their membranes. At least three subpopulations could be distinguished: those that lack Ia antigens and predominate in peritoneal exudate; cells bearing I-A antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-C antigens that are a minor population in both spleen and peritoneum. Internal radioisotope labeling studies confirmed that the I region molecules are synthesized by the macrophages. It is suggested that these different macrophage subpopulations may play distinct roles in the immune response.     

Resiquimod and polyinosinic–polycytidylic acid formulation with aluminum hydroxide as an adjuvant for foot-and-mouth disease vaccine

Background

Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)₃).

Results

Antibody titers to FMDV and CD8⁺ T cells were markedly enhanced in mice immunized to 146S FMDV?+?Al(OH)₃?+?R848?+?poly(I:C) compared with mice immunized to FMDV?+?ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)₃ together biased the immune response toward a Th1-type direction.

Conclusions

These results indicated that the R848 and poly(I:C) together with Al(OH)₃ enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.     

Immunogenetic analysis of tolerance induction in anti-alloantigen delayed type hypersensitivity responses by portal venous pre-inoculation with allogeneic cells

The present study investigates some of the immunogenetic bases for tolerance of anti-allo-delayed type hypersensitivity (DTH) responses as induced by pre-inoculating allogeneic cells via portal venous (p.v.) route. BALB/c mice were injected with totally allogeneic C57BL/6 or H-2 incompatible BALB.B spleen cells via p.v. route. These mice not only failed to exhibit anti-H-2b DTH responses, but also abrogated the potential to generate H-2b-specific DTH responses as induced by the subsequent immunization with H-2b spleen cells via subcutaneous (s.c.) route. The p.v. presensitization with allogeneic spleen cells differing at either class I or class II of major histocompatibility complex (MHC) resulted in the tolerance induction of DTH responses to the respective allogeneic class I or class II MHC antigens. Moreover, the p.v. administration of the class I-positive allogeneic cell fraction depleted of class II-positive component into recipients differing at both class I and class II was capable of inducing anti-class I DTH tolerance. These results indicate that anti-allo-class I or class II DTH tolerance can be induced independently and that the existence of class II antigens on p.v.-presensitized cells is not necessarily required for the tolerance induction of anti-allo-class I DTH response.     

Lactoferrin acts on I-A and I-E/C antigen+ subpopulations of mouse peritoneal macrophages in the absence of T lymphocytes and other cell types to inhibit production of granulocyte-macrophage colony stimulatory factors in vitro

The relationship between Ia antigens on mouse resident peritoneal macrophages and the ability of lactoferrin (LF) to inhibit the production of granulocyte-macrophage colony stimulatory factors (GM-CSF) from these cells was investigated. Detection of the suppressive influence of LF on release of GM-CSF from greater than or equal to 10(5) macrophages/ml/plate required that the conditioned media being assessed for GM-CSF be prepared in the presence of indomethacin and/or be preincubated with anti-ferritin antiserum to respectively stop production of E-type prostaglandins and to remove acidic isoferritin-inhibitory activities that can mask the effects of LF. Treatment of mouse macrophages with monoclonal antibodies to the I-A and I-E/C subregions of Ia antigens in a complement C-dependent cytotoxicity assay killed less than 15% of the cells, but removed all Ia antigen+ macrophages and reduced GM-CSF production by approximately 50%. LF decreased GM-CSF production by untreated macrophages by approximately 50%, but had no effect on macrophages insensitive to treatment with anti-Ia plus C. Macrophages left at 37 degrees C for 5 and 24 hr were not killed by treatment with monoclonal anti-Ia plus C and GM-CSF production by these macrophages was not suppressed by LF. Treatment of macrophages with monoclonal anti-H-2K or anti-Mac-1 plus C reduced GM-CSF production greater than 95%. Anti-I-A, -I-E/C, -H-2K, or -Mac-1, in the absence of C, had no effect on viability of macrophages or on production of GM-CSF, but anti-I-A and -I-E/C each blocked the inhibitory action of LF. Lower concentrations of these antibodies could block the action of LF when anti-I-A and anti-I-E/C were mixed together better than when they were each used separately. The removal of Thy-1.2+ cells from unseparated or adherent peritoneal cells resulted in populations of cells that were up to 100% positive for nonspecific esterase, and did not influence GM-CSF production from these cells, the reduction of GM-CSF from these cells by LF, or the reduction of GM-CSF by the removal of Ia antigen+ cells. The results were similar whether or not T cells were removed from the assay marrow by treatment with antibodies Ly-1.1, Ly-2.2, and Qa4 plus C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of macrophage Ia expression in neonatal mice--role of a splenic suppressor cell

The control of macrophage expression of I region-associated antigens (Ia) in neonatal mice was studied by comparing responses of neonatal and adult mice to immune vs nonimmune stimuli. Adults generated peritoneal exudates rich in Ia-bearing macrophages in response to i.p. injection of live Listeria monocytogenes, Listeria-immune T cells, and heat-killed Listeria, or a soluble mediator termed macrophage Ia-recruiting factor (MIRF). Neonates failed to respond to these stimuli. In contrast, both neonates and adults generated Ia-negative peritoneal exudates when stimulated with thioglycollate. A neonatal spleen cell that blocked the response of adults both to immune T cells and heat-killed Listeria and to MIRF was identified and characterized. Some of the suppressor cells appeared to be early precursors of the phagocytic lineage that develop into mature monocyte-macrophages. Suppression was apparently mediated by metabolites of arachidonic acid since indomethacin and aspirin in vivo blocked the effect. Similar suppressor activity was found in adult bone marrow and in adult resident peritoneal exudate cells. Thus, the phagocytic line autoregulates its surface expression of Ia in both neonatal and adult mice. This mechanism becomes particularly pointed during early development and could contribute to the lack of immunity during ontogeny.     

Induction of virus-neutralizing antibodies by bacteria expressing the C3 poliovirus epitope in the periplasm. The route of immunization influences the isotypic distribution and the biologic activity of the antipoliovirus antibodies

Two viral epitopes (C3 neutralization epitope from poliovirus type 1 and the 132-145 peptide from the PreS2 region from hepatitis B virus) have been expressed in the Escherichia coli periplasm as protein fusion with the maltose binding protein (MalE protein). Immunization of mice with live bacteria expressing the foreign viral epitopes in their periplasm elicited high antibody titers against the viral peptide as well as against the corresponding virus. This demonstrates for the first time in the case of defined epitopes that, when live bacteria are used as immunogens, presentation at the cell surface is not a prerequisite to obtain an antibody response. On the other hand, the induction of antiviral antibody responses by these recombinant bacteria depended dramatically on the route of immunization: a response was induced by live bacteria through the i.v. route but not through the s.c. route. However, when bacteria were heat killed or when the MalE hybrid protein was released under a soluble form from the cell, a response was induced even upon s.c. immunization. From these results, we suggest that in order to induce high levels of antibodies by the s.c. route, a major parameter for bacterial Ag would be their capacity to be released into a soluble form before the interaction of the bacteria with the APC. This would permit the presentation by B cells rather than by phagocytic cells. Finally, we demonstrate that the route of immunization influences the isotypic distribution and the neutralizing activity of the antipoliovirus antibodies. Such results may have major implications for the development of bacterial vaccines based on fusion proteins.     

Effect of pertussis vaccine on the functional activity of the peritoneal and splenic macrophages in 2 mouse strains genetically differing by the intensity of their immune response

The immunostimulating effect of corpuscular pertussis vaccine on the antigen-presenting and bactericidal functions of peritoneal and splenic macrophages in CBA and C57BL/6 mice, differing in the intensity of immune response to sheep red blood cells and Salmonella typhimurium, has been studied. The study has revealed that the injection of pertussis vaccine alters the functional activity of the cells under study, the effect depending on the immunizing dose, the strain of mice and the time elapsed from the moment of immunization. Pertussis vaccine enhances the low capacity of macrophages for antigen presentation in C57BL/6 mice with low responsiveness and alters the resistance of peritoneal and splenic macrophages to the cytopathic action of salmonellae.     

Secondary cell-mediated cytotoxic response to syngeneic mouse tumor challenge.

When C57BL/6 mice previously immunized with murine sarcoma virus (MSV) were challenged with a Rauscher virus-induced lymphoma, RBL-5, a secondary cell-mediated cytotoxic response could be detected by the Cr release cytotoxicity assay. The level and distribution of the secondary cytotoxic response was affected by the route of challenge. Animals injected i.p. demonstrated a high level of cytotoxicity in the peritoneal exudate cells 3 days after challenge and subsequently cytotoxicity was detected in most lymphoid organs, although at lower levels. However, when the animals were challenged intramuscularly in the leg, the response was not detected as rapidly and furthermore cytotoxic lymphocytes were found only in the draining lymph node and not in other lymphoid organs. Treatment of the effector cells with anti-theta and complement showed the secondary response to be predominately dependent on T cells. In addition, the cytotoxicity was specific in that cells lacking cross-reacting antigens were not killed by these attacker cells from mice undergoing a secondary response.