Sera Of Vaccinated Mice Research Articles

The Immunogenic Potential of PCSK9 Peptide Vaccine in Mice.

To evaluate the immunogenic potential of the carrier-free peptide-based anti-PCSK9 (proprotein convertase subtilisin/kexin 9) vaccine in albino mice. The immunogenic pcsk9 peptide and 0.4% alum adjuvant were mixed thoroughly at a 1:1 ratio and used as a vaccine formulation. To assess the humoral immune response, animals' blood was sampled two weeks after the last immunization. The ELISA method was employed to measure serum anti-PCSK9 antibody titers, PCSK9 concentrations, and PCSK9/LDLR interaction. ELISA analysis showed significant induction of IgG antibody titers by PCSK9 peptide vaccine in vaccinated mice sera compared to the control mice (in male and female mice were 12000±586 and 11566±642, respectively, p<0.001). Mechanistic analyses showed a significant reduction in serum PCSK9 concentrations by vaccine-induced antibodies in vaccine groups compared to the control groups (in male mice by 29±5 ng/mL (22.4%), p<0.001 and female mice by 26±5 ng / mL (21.0%), p<0.001). Serum concentrations of PCSK9 in control and vaccine groups were 131±8.6 ng / mL and 102±8.1 ng/ml in male mice and 124±6 ng/ml and 98±10 ng/ml in female mice, respectively. Moreover, vaccine-induced antibodies inhibited the PCSK9-LDLR interaction in male and female groups by 34% and 26%, respectively. No significant difference was detected between the male and female groups in all tests (p>0.05). According to our results, the PCSK9 peptide vaccine provoked the humoral immune system in albino mice to produce functional antibodies that inhibit plasma PCSK9. These effects were seen in both genders without any significant difference.

Development of SARS-CoV-2 neutralizing antibody detection assay by using recombinant plant-produced proteins

Detecting immunity against SARS-CoV-2 is vital for evaluating vaccine response and natural infection, but conventional virus neutralization test (cVNT) requires BSL3 and live viruses, and pseudo-virus neutralization test (pVNT) needs specialized equipment and trained professionals. The surrogate virus neutralization test (sVNT) was developed to overcome these limitations. This study explored the use of angiotensin converting enzyme 2 (ACE2) produced from Nicotiana benthamiana for the development of an affordable neutralizing antibodies detection assay. The results showed that the plant-produced ACE2 can bind to the receptor binding domain (RBD) of the SARS-CoV-2, and was used to develop sVNT with plant-produced RBD protein. The sVNT developed using plant-produced proteins showed high sensitivity and specificity when validated with a group of 30 RBD vaccinated mice sera and the results were correlated with cVNT titer. This preliminary finding suggests that the plants could offer a cost-effective platform for producing diagnostic reagents.

Induction of Protective Immunity against Toxoplasmosis in BALB/c Mice Vaccinated with Toxoplasma gondii Rhoptry-1.

Toxoplasma gondii is the causative agent for toxoplasmosis. The rhoptry protein 1 (ROP1) is secreted by rhoptry, an apical secretory organelle of the parasite. ROP1 plays an important role in host cell invasion. In this study, the efficacy of ROP1 as a vaccine candidate against toxoplasmosis was evaluated through intramuscular or subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated) and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Briefly, a recombinant DNA plasmid (pVAX1-GFP-ROP1) was expressed in CHO cells while expression of recombinant ROP1 protein (rROP1) was carried out in Escherichia coli expression system. Immunization study involved injection of the recombinant pVAX1-ROP1 and purified rROP1 into different group of mice. Empty vector and PBS served as two different types of negative controls. Results obtained demonstrated that ROP1 is an immunogenic antigen that induced humoral immune response whereby detection of a protein band with expected size of 43 kDa was observed against vaccinated mice sera through western blot analysis. ROP1 antigen was shown to elicit cellular-mediated immunity as well whereby stimulated splenocytes with total lysate antigen (TLA) and rROP1 from pVAX1-ROP1 and rROP1-immunized mice, respectively, readily proliferated and secreted large amount of IFN-γ (712 ± 28.1 pg/ml and 1457 ± 31.19 pg/ml, respectively) and relatively low IL-4 level (94 ± 14.5 pg/ml and 186 ± 14.17 pg/ml, respectively). These phenomena suggested that Th1-favored immunity was being induced. Vaccination with ROP1 antigen was able to provide partial protection in the vaccinated mice against lethal challenge with virulent RH strain of tachyzoites. These findings proposed that the ROP1 antigen is a potential candidate for the development of vaccine against toxoplasmosis.

Abstract 2560: Strong cross-reactive responses to pancreatic cancer cells induced by α-gal epitopes-expressing tumor lysate and their implications for a universal cancer vaccine

Abstract We investigated the effects of whole cell vaccination with α-gal epitopes-expressing pancreatic cancer (PC) cells (Cancer Res; 70 (13); 5259-69, 2010) and examined the usefulness of this vaccine in the induction of specific anti-tumor-responses and improvement of survival. But, these effects were somewhat weak. We hypothesized that tumor lysate (t-ly), which contains many tumor-associated antigens (TAAs) of both PC and stromal cells is a suitable vaccination material and it can elicit a broadly protective anti-tumor responses. In this study, we investigate that vaccination with α-gal epitopes-expressing PC t-ly can induce cross-reactive protections to heterosubtypic PC cells. Human PC cell lines, BxPC3 (Bx), expressing Mesothelin, and MiaPaCa (Mia), expressing MUC1 were employed. PC cell lines were injected s.c. into NOD/SCID mice. The grown tumors were enucleated and homogenized for making t-ly. To synthesize α-gal epitopes on t-ly, we cloned the α1,3 galactosyltransferase (α1,3GT) cDNA from a New World monkey and isolated a soluble form of enzyme in the yeast expression system of Pichia pastoris. These t-lys were incubated with UDP-Gal and α1,3GT. α1,3GT KO mice were immunized with pig tissues to produce anti-Gal Ab. The high anti-Gal KO mice were vaccinated intraperitoneally by either parental (control group; Bx-t-ly or Mia-t-ly) or α-gal-t-ly (α-gal group; α-gal-Bx-t-ly or α-gal-Mia-t-ly). No expression of α-gal epitopes were observed in parental t-lys, whereas high expression levels were clearly detected on α-gal-t-lys. α-gal-Bx-t-ly vaccination elicited strong responses of anti-Bx IgG and anti-Mesothelin IgG, whereas Bx-t-ly vaccination did not induce such Ab responses. For Mia-t-ly vaccination, KO mice of α-gal group clearly induce anti-Mia IgG and anti-MUC1 IgG, but no Ab responses were observed in control group. Productions of either anti-Bx or anti-Mia IgG in α-gal group were 64-fold higher than those in control group. α-gal-Bx-t-ly vaccinated KO mice abundantly produced cross-reactive IgG Abs to Mia cells, whereas no cross-reactive humoral responses to Mia cells were induced by Bx-t-ly vaccine. Vaccinated mice sera from α-gal-Mia-t-ly strongly cross-react to Bx cells. To demonstrate in vivo tumor destruction, splenocytes from vaccinated KO mice were prepared, and these splenocytes were inoculated i.p. into NOD/SCID mice. Either live Bx or Mia cells were challenged with s.c. injection. Both Bx and Mia tumors in mice inoculated from parental group reached the size of 100 mm2 at approximately 22 days after injection. For tumors in mice transferred from α-gal group, no regrowth tumors were identified. We conclude that α-gal epitopes-expressing t-ly vaccine can elicit durable and broadly protective immune response to subtypic PC cells, and that such vaccination may provide implications for a universal cancer vaccine to cure for the patients with PC. Citation Format: Masahiro Tanemura, Eiji Miyoshi, Hiroaki Nagano, Masafumi Inoue, Toshimitsu Irei, Shinya Yamashita, Kenta Furukawa, Katsuyoshi Matsunami, Wataru Kamiike, Masaki Mori, Yuichiro Doki. Strong cross-reactive responses to pancreatic cancer cells induced by α-gal epitopes-expressing tumor lysate and their implications for a universal cancer vaccine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2560. doi:10.1158/1538-7445.AM2014-2560

Immunization of Mice with Recombinant Mosquito Salivary Protein D7 Enhances Mortality from Subsequent West Nile Virus Infection via Mosquito Bite

BackgroundMosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.Methodology/Principal FindingsWe identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.Conclusions/SignificanceVector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.

Immunoproteomic analysis of the protective response obtained from vaccination with Candida albicans ecm33 cell wall mutant in mice

Systemic candidiasis remains a major cause of disease and death, particularly among immunocompromised patients. The cell wall of Candida albicans defines the interface between host and pathogen and surface proteins are major elicitors of host immune responses during candidiasis. The C. albicans ecm33 mutant (RML2U) presents an altered cell wall, which entails an increase in the outermost protein layer. Vaccination of BALB/c mice with RML2U mutant protected them from a subsequent lethal infection with virulent strain SC5314 in a systemic candidiasis model. Using immunoproteomics (2-DE followed by Immunoblotting) we detected 29 immunoreactive proteins specifically recognized by antibodies from vaccinated mice sera, six of which are described as immunogenic for the first time (Gnd1p, Cit1p, Rpl10Ep, Yst1p, Cys4p, Efb1p). Furthermore, identification of wild type and mutant cell surface proteome (surfome), confirmed us that the mutant surfome presented a larger number of proteins than the wild type. Interestingly, proteins exclusively identified in the mutant surfome (Met6p, Eft2p, Tkl1p, Rpl10Ep, Atp1p, Atp2p) were also detected as immunogenic, supporting the idea that their surface location enhances their immunoprotective capacity.

Immunogenecity of synthetic peptides representing linear B-cell epitopes of VapA of Rhodococcus equi

Amino acid 65–78 of membrane protein VapA of the facultative intracellular Rhodococcus equi contained an immunodominant N-terminal B-cell epitope (N15Y peptide). Safety and immunogenecity of a synthetic peptide consisting of the amino acid 65–78 of VapA (peptide N15Y) were evaluated first in mice and in healthy adult horses. A single dose of a peptide-VapA vaccine induced and only in presence of adjuvant, specific IgG antibodies in sera of mice. After challenge with virulent R. equi 3 weeks after immunization, tissue clearance was more delayed in immunized mice than in control mice. An antibody-mediated response (restricted to IgG1 and IgG2b subclasses) predominated in vaccinated mice sera and no specific lymphocytes proliferation was observed. Next, a total of 15 mares were given systemic inoculation of N15Y peptide with IMS3012 ( n=4) or IMS2211 ( n=4) or ISA35 ( n=4) or placebo ( n=2). Serological responses to the peptide vaccine were found in all but not in placebo group. A significant increase of IgGb subclass in sera of vaccinated mare with N15Y peptide in presence of IMS3012 was observed in comparison to IMS2211 or ISA35 or control group. Moreover, INF-γ, IL-2 and IL-10 mRNA expression increased more significantly in peripheral blood lymphocytes of IMS3012 group than in IMS2211 or ISA35 group. Interestingly, a significant decrease of IL-4 mRNA expression (undetectable level) was observed with all adjuvants. These results support the use of peptide N15Y in presence of IMS3012 adjuvant in future studies of protection of foals against R. equi.