Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses... more Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses have focused on targeting individual viruses, although their geographical and bat reservoir host overlaps warrant creation of multivalent vaccines. Here we explored whether replication-incompetent pseudotyped vesicular stomatitis virus (VSV) virions or NiV-based virus-like particles (VLPs) were suitable multivalent vaccine platforms by co-incorporating multiple surface glycoproteins from NiV, HeV, and EBOV onto these virions. We then enhanced the vaccines’ thermotolerance using carbohydrates to enhance applicability in global regions that lack cold-chain infrastructure. Excitingly, in a Syrian hamster model of disease, the VSV multivalent vaccine elicited safe, strong, and protective neutralizing antibody responses against challenge with NiV, HeV, or EBOV. Our study provides proof-of-principle evidence that replication-incompetent multivalent viral particle vaccines are sufficient to pr...
The potential for future coronavirus outbreaks highlights the need to develop strategies and tool... more The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2’ cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2’ cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutral...
Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) com... more Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mi...
The quest for vaccine development against Chlamydia trachomatis (CT), the leading cause of bacter... more The quest for vaccine development against Chlamydia trachomatis (CT), the leading cause of bacterial sexually transmitted diseases worldwide, has escalated to reduce its occurrence and global economic burden. Recently, nanovaccines have emerged as attractive candidates due to flexibility in their formulation, biodegradability, biocompatibility and adjuvant properties. Nanovaccines applicability depends on their physical properties for their immunogenicity, which is vital for biosafety and therapeutic efficacy studies. We have developed a nanovaccine against CT employing its recombinant MOMP (major outer membrane protein) by encapsulating it within PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles. Physiostructural characterizations included Zeta-sizing, Zetapotential, DSC (Differential Scanning Calorimetry), UV spectroscopy and in vitro release of rMOMP. Encapsulated rMOMP nanoparticles were small (~200 nm), thermally stable (90oC) and negatively charged (-13 mV). UV spe...
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was cle... more As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptab...
Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chla... more Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chlamydial infections. The anti-inflammatory cytokine, interleukin (IL)-10, has been reported to efficiently suppress the secretion of inflammatory cytokines triggered by Chlamydia in mouse macrophages. Though IL-10 is employed in clinical applications, its therapeutic usage is limited due to its short half-life. Here, we document the successful encapsulation of IL-10 within the biodegradable polymeric nanoparticles of PLA-PEG (Poly (lactic acid)-Poly (ethylene glycol), to prolong its half-life. Our results show the encapsulated-IL-10 size (~238 nm), zeta potential (−14.2 mV), polydispersity index (0.256), encapsulation efficiency (~77%), and a prolonged slow release pattern up to 60 days. Temperature stability of encapsulated-IL-10 was favorable, demonstrating a heat capacity of up to 89 °C as shown by differential scanning calorimetry analysis. Encapsulated-IL-10 modulated the release of I...
Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (... more Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of )] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate -specific CD4 T-cell immune effector responses. In the present study, we employed the mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced -specific adaptive immune responses predominated by IFN-γ along with CD4 T-cells proliferation and their differentiation to CD4 memory (CD44 CD62L) and effector (CD44 CD62L) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific seru...
We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M... more We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracell...
There is a persisting global burden and considerable public health challenge by the plethora of o... more There is a persisting global burden and considerable public health challenge by the plethora of ocular, genital and respiratory diseases caused by members of the Gram-negative bacteria of the genus Chlamydia. The major diseases are conjunctivitis and blinding trachoma, non-gonococcal urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and interstitial pneumonia. The failures in screening and other prevention programs led to the current medical opinion that an efficacious prophylactic vaccine is the best approach to protect humans from chlamydial infections. Unfortunately, there is no human Chlamydia vaccine despite successful veterinary vaccines. A major challenge has been the effective delivery of vaccine antigens to induce safe and effective immune effectors to confer long-term protective immunity. The dawn of the era of biodegradable polymeric nanoparticles and the adjuvanted derivatives may accelerate the realization of the dream of ...
The repair or replacement of damaged skins is still an important, challenging public health probl... more The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolera...
Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial c... more Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulat...
Nanomedicine: Nanotechnology, Biology and Medicine, 2016
Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children ... more Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children and older adults. The use of metallic nanoparticles as potential therapeutics is being explored against respiratory viruses like Influenza, Parainfluenza and Adenovirus. In this study, we showed that gold nanorods (GNRs) inhibit RSV in HEp-2 cells and BALB/c mice by 82% and 56%, respectively. The RSV inhibition correlated with marked upregulated antiviral genes due to GNR mediated TLR, NOD-like receptor and RIG-I-like receptor signaling pathways. Transmission electron microscopy of lungs showed GNRs in the endocytotic vesicles and histological analyses indicated infiltration by neutrophils, eosinophils and monocytes correlating with clearance of RSV. In addition, production of cytokines and chemokines in the lungs indicates recruitment of immune cells to counter RSV replication. To our knowledge, this is the first in vitro and in vivo report that provides possible antiviral mechanisms of GNRs against RSV.
Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses... more Experimental vaccines for the deadly zoonotic Nipah (NiV), Hendra (HeV), and Ebola (EBOV) viruses have focused on targeting individual viruses, although their geographical and bat reservoir host overlaps warrant creation of multivalent vaccines. Here we explored whether replication-incompetent pseudotyped vesicular stomatitis virus (VSV) virions or NiV-based virus-like particles (VLPs) were suitable multivalent vaccine platforms by co-incorporating multiple surface glycoproteins from NiV, HeV, and EBOV onto these virions. We then enhanced the vaccines’ thermotolerance using carbohydrates to enhance applicability in global regions that lack cold-chain infrastructure. Excitingly, in a Syrian hamster model of disease, the VSV multivalent vaccine elicited safe, strong, and protective neutralizing antibody responses against challenge with NiV, HeV, or EBOV. Our study provides proof-of-principle evidence that replication-incompetent multivalent viral particle vaccines are sufficient to pr...
The potential for future coronavirus outbreaks highlights the need to develop strategies and tool... more The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2’ cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2’ cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutral...
Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) com... more Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mi...
The quest for vaccine development against Chlamydia trachomatis (CT), the leading cause of bacter... more The quest for vaccine development against Chlamydia trachomatis (CT), the leading cause of bacterial sexually transmitted diseases worldwide, has escalated to reduce its occurrence and global economic burden. Recently, nanovaccines have emerged as attractive candidates due to flexibility in their formulation, biodegradability, biocompatibility and adjuvant properties. Nanovaccines applicability depends on their physical properties for their immunogenicity, which is vital for biosafety and therapeutic efficacy studies. We have developed a nanovaccine against CT employing its recombinant MOMP (major outer membrane protein) by encapsulating it within PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles. Physiostructural characterizations included Zeta-sizing, Zetapotential, DSC (Differential Scanning Calorimetry), UV spectroscopy and in vitro release of rMOMP. Encapsulated rMOMP nanoparticles were small (~200 nm), thermally stable (90oC) and negatively charged (-13 mV). UV spe...
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was cle... more As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic expanded, it was clear that effective testing for the presence of neutralizing antibodies in the blood of convalescent patients would be critical for development of plasma-based therapeutic approaches. To address the need for a high-quality neutralization assay against SARS-CoV-2, a previously established fluorescence reduction neutralization assay (FRNA) against Middle East respiratory syndrome coronavirus (MERS-CoV) was modified and optimized. The SARS-CoV-2 FRNA provides a quantitative assessment of a large number of infected cells through use of a high-content imaging system. Because of this approach, and the fact that it does not involve subjective interpretation, this assay is more efficient and more accurate than other neutralization assays. In addition, the ability to set robust acceptance criteria for individual plates and specific test wells provided further rigor to this assay. Such agile adaptab...
Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chla... more Inflammation, as induced by the presence of cytokines and chemokines, is an integral part of chlamydial infections. The anti-inflammatory cytokine, interleukin (IL)-10, has been reported to efficiently suppress the secretion of inflammatory cytokines triggered by Chlamydia in mouse macrophages. Though IL-10 is employed in clinical applications, its therapeutic usage is limited due to its short half-life. Here, we document the successful encapsulation of IL-10 within the biodegradable polymeric nanoparticles of PLA-PEG (Poly (lactic acid)-Poly (ethylene glycol), to prolong its half-life. Our results show the encapsulated-IL-10 size (~238 nm), zeta potential (−14.2 mV), polydispersity index (0.256), encapsulation efficiency (~77%), and a prolonged slow release pattern up to 60 days. Temperature stability of encapsulated-IL-10 was favorable, demonstrating a heat capacity of up to 89 °C as shown by differential scanning calorimetry analysis. Encapsulated-IL-10 modulated the release of I...
Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (... more Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of )] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate -specific CD4 T-cell immune effector responses. In the present study, we employed the mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced -specific adaptive immune responses predominated by IFN-γ along with CD4 T-cells proliferation and their differentiation to CD4 memory (CD44 CD62L) and effector (CD44 CD62L) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific seru...
We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M... more We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracell...
There is a persisting global burden and considerable public health challenge by the plethora of o... more There is a persisting global burden and considerable public health challenge by the plethora of ocular, genital and respiratory diseases caused by members of the Gram-negative bacteria of the genus Chlamydia. The major diseases are conjunctivitis and blinding trachoma, non-gonococcal urethritis, cervicitis, pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and interstitial pneumonia. The failures in screening and other prevention programs led to the current medical opinion that an efficacious prophylactic vaccine is the best approach to protect humans from chlamydial infections. Unfortunately, there is no human Chlamydia vaccine despite successful veterinary vaccines. A major challenge has been the effective delivery of vaccine antigens to induce safe and effective immune effectors to confer long-term protective immunity. The dawn of the era of biodegradable polymeric nanoparticles and the adjuvanted derivatives may accelerate the realization of the dream of ...
The repair or replacement of damaged skins is still an important, challenging public health probl... more The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolera...
Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial c... more Pseudomonas aeruginosa infection induces vigorous inflammatory mediators secreted by epithelial cells, which do not necessarily eradicate the pathogen. Nonetheless, it reduces lung function due to significant airway damage, most importantly in cystic fibrosis patients. Recently, we published that TP359, a proprietary cationic peptide had potent bactericidal effects against P. aeruginosa, which were mediated by down-regulating its outer membrane biogenesis genes. Herein, we hypothesized that TP359 bactericidal effects could also serve to regulate P. aeruginosa-induced lung inflammation. We explored this hypothesis by infecting human A549 lung cells with live P. aeruginosa non-isogenic, mucoid and non-mucoid strains and assessed the capacity of TP359 to regulate the levels of elicited TNFα, IL-6 and IL-8 inflammatory cytokines. In all instances, the mucoid strain elicited higher concentrations of cytokines in comparison to the non-mucoid strain, and TP359 dose-dependently down-regulat...
Nanomedicine: Nanotechnology, Biology and Medicine, 2016
Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children ... more Respiratory syncytial virus (RSV) causes severe pneumonia and bronchiolitis in infants, children and older adults. The use of metallic nanoparticles as potential therapeutics is being explored against respiratory viruses like Influenza, Parainfluenza and Adenovirus. In this study, we showed that gold nanorods (GNRs) inhibit RSV in HEp-2 cells and BALB/c mice by 82% and 56%, respectively. The RSV inhibition correlated with marked upregulated antiviral genes due to GNR mediated TLR, NOD-like receptor and RIG-I-like receptor signaling pathways. Transmission electron microscopy of lungs showed GNRs in the endocytotic vesicles and histological analyses indicated infiltration by neutrophils, eosinophils and monocytes correlating with clearance of RSV. In addition, production of cytokines and chemokines in the lungs indicates recruitment of immune cells to counter RSV replication. To our knowledge, this is the first in vitro and in vivo report that provides possible antiviral mechanisms of GNRs against RSV.
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Papers by Saurabh Dixit