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1.
Proc Natl Acad Sci U S A ; 120(1): e2217883120, 2023 Jan 03.
Article in English | MEDLINE | ID: mdl-36574685

ABSTRACT

Antibody heavy chain (HC) and light chain (LC) variable region exons are assembled by V(D)J recombination. V(D)J junctional regions encode complementarity-determining-region 3 (CDR3), an antigen-contact region immensely diversified through nontemplated nucleotide additions ("N-regions") by terminal deoxynucleotidyl transferase (TdT). HIV-1 vaccine strategies seek to elicit human HIV-1 broadly neutralizing antibodies (bnAbs), such as the potent CD4-binding site VRC01-class bnAbs. Mice with primary B cells that express receptors (BCRs) representing bnAb precursors are used as vaccination models. VRC01-class bnAbs uniformly use human HC VH1-2 and commonly use human LCs Vκ3-20 or Vκ1-33 associated with an exceptionally short 5-amino-acid (5-aa) CDR3. Prior VRC01-class models had nonphysiological precursor levels and/or limited precursor diversity. Here, we describe VRC01-class rearranging mice that generate more physiological primary VRC01-class BCR repertoires via rearrangement of VH1-2, as well as Vκ1-33 and/or Vκ3-20 in association with diverse CDR3s. Human-like TdT expression in mouse precursor B cells increased LC CDR3 length and diversity and also promoted the generation of shorter LC CDR3s via N-region suppression of dominant microhomology-mediated Vκ-to-Jκ joins. Priming immunization with eOD-GT8 60mer, which strongly engages VRC01 precursors, induced robust VRC01-class germinal center B cell responses. Vκ3-20-based responses were enhanced by N-region addition, which generates Vκ3-20-to-Jκ junctional sequence combinations that encode VRC01-class 5-aa CDR3s with a critical E residue. VRC01-class-rearranging models should facilitate further evaluation of VRC01-class prime and boost immunogens. These new VRC01-class mouse models establish a prototype for the generation of vaccine-testing mouse models for other HIV-1 bnAb lineages that employ different HC or LC Vs.


Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , Vaccines , Mice , Humans , Animals , Broadly Neutralizing Antibodies , Antibodies, Neutralizing , HIV-1/genetics , HIV Antibodies , DNA Nucleotidylexotransferase , Complementarity Determining Regions/genetics , HIV Infections/prevention & control
2.
Int J Mol Sci ; 23(20)2022 Oct 12.
Article in English | MEDLINE | ID: mdl-36292999

ABSTRACT

Bacteriophages (phages), as natural antibacterial agents, are being rediscovered because of the growing threat of multi- and pan-drug-resistant bacterial pathogens globally. However, with an estimated 1031 phages on the planet, finding the right phage to recognize a specific bacterial host is like looking for a needle in a trillion haystacks. The host range of a phage is primarily determined by phage tail fibers (or spikes), which initially mediate reversible and specific recognition and adsorption by susceptible bacteria. Recent significant advances at single-molecule and atomic levels have begun to unravel the structural organization of tail fibers and underlying mechanisms of phage-host interactions. Here, we discuss the molecular mechanisms and models of the tail fibers of the well-characterized T4 phage's interaction with host surface receptors. Structure-function knowledge of tail fibers will pave the way for reprogramming phage host range and will bring future benefits through more-effective phage therapy in medicine. Furthermore, the design strategies of tail fiber engineering are briefly summarized, including machine-learning-assisted engineering inspired by the increasingly enormous amount of phage genetic information.


Subject(s)
Bacteriophages , Bacteriophages/physiology , Host Specificity , Virion , Carrier Proteins , Anti-Bacterial Agents
3.
Sci Immunol ; 7(76): eadd5446, 2022 10 28.
Article in English | MEDLINE | ID: mdl-35951767

ABSTRACT

SARS-CoV-2 Omicron subvariants have generated a worldwide health crisis due to resistance to most approved SARS-CoV-2 neutralizing antibodies and evasion of vaccination-induced antibodies. To manage Omicron subvariants and prepare for new ones, additional means of isolating broad and potent humanized SARS-CoV-2 neutralizing antibodies are desirable. Here, we describe a mouse model in which the primary B cell receptor (BCR) repertoire is generated solely through V(D)J recombination of a human VH1-2 heavy chain (HC) and, substantially, a human Vκ1-33 light chain (LC). Thus, primary humanized BCR repertoire diversity in these mice derives from immensely diverse HC and LC antigen-contact CDR3 sequences generated by nontemplated junctional modifications during V(D)J recombination. Immunizing this mouse model with SARS-CoV-2 (Wuhan-Hu-1) spike protein immunogens elicited several VH1-2/Vκ1-33-based neutralizing antibodies that bound RBD in a different mode from each other and from those of many prior patient-derived VH1-2-based neutralizing antibodies. Of these, SP1-77 potently and broadly neutralized all SARS-CoV-2 variants through BA.5. Cryo-EM studies revealed that SP1-77 bound RBD away from the receptor-binding motif via a CDR3-dominated recognition mode. Lattice light-sheet microscopy-based studies showed that SP1-77 did not block ACE2-mediated viral attachment or endocytosis but rather blocked viral-host membrane fusion. The broad and potent SP1-77 neutralization activity and nontraditional mechanism of action suggest that it might have therapeutic potential. Likewise, the SP1-77 binding epitope may inform vaccine strategies. Last, the type of humanized mouse models that we have described may contribute to identifying therapeutic antibodies against future SARS-CoV-2 variants and other pathogens.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Mice , Animals , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2 , Membrane Fusion , Antibodies, Viral , Antibodies, Neutralizing , Epitopes , Receptors, Antigen, B-Cell
4.
mBio ; 13(4): e0182222, 2022 08 30.
Article in English | MEDLINE | ID: mdl-35900097

ABSTRACT

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.


Subject(s)
Bacteriophages , COVID-19 , Adjuvants, Immunologic , Animals , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Mice , Mice, Inbred BALB C , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
5.
Vaccines (Basel) ; 10(6)2022 May 24.
Article in English | MEDLINE | ID: mdl-35746442

ABSTRACT

Dengue virus and Zika virus are mosquito-borne, single-stranded, positive-sense RNA viruses that belong to the Flaviviridae family. Both the viruses are closely related and have similarities with other flaviviruses. Dengue virus (DENV) causes a severe febrile illness with fever, joint pain, and rash leading to a life-threatening condition in severe cases. While Zika virus (ZIKV) primarily causes mild fever, it can be passed from a pregnant mother to her fetus, resulting in severe birth defect microcephaly and even causing a rare autoimmune disease-Guillain-Barre syndrome. To date, there are no approved DENV and ZIKA vaccines available, except a Dengue vaccine (Dengvaxia, Sanofi Pasteur Inc., Lyon, France) recently approved to be used only for 9-16 years of age groups living in endemic areas and having a previous record of confirmed dengue infection. There are several potential vaccine candidates in the clinical trials based on multiple vaccine platforms, such as live attenuated, subunit, nucleic acid, and viral vector-based vaccines. In the current review, we have focused exclusively on the nucleic acid vaccine platform and discussed the progress of all the DNA/RNA vaccine candidates under preclinical and clinical studies for DENV and ZIKA viruses. Additionally, we have described a brief history of the emergence of these flaviviruses, major structural similarities between them, prominent vaccine targets, and the mechanism of virus entry and infection.

6.
Sci Adv ; 7(37): eabh1547, 2021 Sep 10.
Article in English | MEDLINE | ID: mdl-34516878

ABSTRACT

A "universal" platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (TH1) and TH2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.

7.
Methods Mol Biol ; 2410: 209-228, 2022.
Article in English | MEDLINE | ID: mdl-34914049

ABSTRACT

The COVID-19 pandemic brought to the fore the urgent need for vaccine design and delivery platforms that can be rapidly deployed for manufacture and distribution. Though the mRNA and adenoviral vector platforms have been enormously successful to control SARS-CoV-2 viral infections, it is unclear if this could be replicated against more complex pathogens or the emerging variants. Recently, we described a "universal" platform that can incorporate multiple vaccine targets into the same nanoparticle scaffold by CRISPR engineering of bacteriophage T4. A T4-COVID vaccine designed with this technology elicited broad immunogenicity and complete protection against virus challenge in a mouse model. Here, we describe the detailed methodology to generate recombinant bacteriophage T4 backbones using CRISPR that can also be broadly applicable to other bacteriophages that abundantly pervade the Earth.


Subject(s)
Bacteriophage T4 , COVID-19 Vaccines , COVID-19 , Clustered Regularly Interspaced Short Palindromic Repeats , Animals , Bacteriophage T4/genetics , COVID-19/prevention & control , Humans , Mice , Pandemics , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vaccine Development
8.
bioRxiv ; 2021 Jan 20.
Article in English | MEDLINE | ID: mdl-33501450

ABSTRACT

A "universal" vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both T H 1 and T H 2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

9.
Vaccines (Basel) ; 8(4)2020 Nov 03.
Article in English | MEDLINE | ID: mdl-33153096

ABSTRACT

With a death toll of over one million worldwide, the COVID-19 pandemic caused by SARS-CoV-2 has become the most devastating humanitarian catastrophe in recent decades. The fear of acquiring infection and spreading to vulnerable people has severely impacted society's socio-economic status. To put an end to this growing number of infections and deaths as well as to switch from restricted to everyday living, an effective vaccine is desperately needed. As a result, enormous efforts have been made globally to develop numerous vaccine candidates in a matter of months. Currently, over 30 vaccine candidates are under assessment in clinical trials, with several undergoing preclinical studies. Here, we reviewed the major vaccine candidates based on the specific vaccine platform utilized to develop them. We also discussed the immune responses generated by these candidates in humans and preclinical models to determine vaccine safety, immunogenicity, and efficacy. Finally, immune responses induced in recovered COVID-19 patients and their possible vaccine development implications were also briefly reviewed.

10.
J Gen Virol ; 101(12): 1289-1299, 2020 12.
Article in English | MEDLINE | ID: mdl-32915123

ABSTRACT

Protective antibody responses to human immunodeficiency virus (HIV)-1 infection evolve only in a fraction of infected individuals by developing broadly neutralizing antibodies (bnAbs) and/or effector functions such as antibody-dependent cellular cytotoxicity (ADCC). HIV-1 chronically infected adults and children on combination antiretroviral therapy (cART) showed a reduction in ADCC activity and improvement in HIV-1 specific neutralizing antibody (nAb) responses. Early initiation of cART in infected adults is found to be beneficial in reducing the viral load and delaying disease progression. Herein, we longitudinally evaluated the effect of cART on HIV-1 specific plasma ADCC and nAb responses in a cohort of 20 perinatally HIV-1 subtype-C infected infants and children ≤2 years of age, pre-cART and up to 1 year post-cART initiation. Significant reductions in HIV-1 specific plasma ADCC responses to subtype-C and subtype-B viruses and improvement in HIV-1 neutralization were observed in HIV-1 infected children 1 year post-cART initiation. A positive correlation between reduction in viral load and the loss of ADCC response was observed. This study provides information aiding the understanding of the effects of early initiation of cART on antibody effector functions and viral neutralization in HIV-1 infected children, which needs to be further evaluated in large cohorts of HIV-1 infected children on cART to plan future intervention strategies.


Subject(s)
Anti-HIV Agents/therapeutic use , Antibody-Dependent Cell Cytotoxicity/drug effects , HIV Antibodies/immunology , HIV Infections/drug therapy , HIV Infections/immunology , HIV-1/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antiretroviral Therapy, Highly Active , Child, Preschool , Cohort Studies , Dideoxynucleosides/therapeutic use , Drug Combinations , HIV Antibodies/blood , HIV Infections/virology , HIV-1/classification , Humans , Infant , Lamivudine/therapeutic use , Lopinavir , Ritonavir , Viral Load , Zidovudine/therapeutic use
11.
Front Microbiol ; 10: 954, 2019.
Article in English | MEDLINE | ID: mdl-31130936

ABSTRACT

Bacteriophages (phages) are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. The therapeutic use of phages to treat bacterial infections ("phage therapy") was conceived by Felix d'Herelle nearly a century ago. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases. In addition to phage therapy, phages have also been developed as vaccine platforms to deliver antigens as part of virus-like nanoparticles that can stimulate immune responses and prevent pathogen infections. Phage engineering promises to generate phage variants with unique properties for prophylactic and therapeutic applications. These approaches have created momentum to accelerate basic as well as translational phage research and potential development of therapeutics in the near future.

12.
Adv Drug Deliv Rev ; 145: 57-72, 2019 05.
Article in English | MEDLINE | ID: mdl-29981801

ABSTRACT

Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host's innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a "universal" VLP platform to design future multivalent vaccines against complex and emerging pathogens.


Subject(s)
Bacteriophage T4 , Communicable Diseases/therapy , Nanoparticles/administration & dosage , Phage Therapy , Vaccines, Virus-Like Particle/administration & dosage , Animals , Communicable Diseases/immunology , Humans
13.
Int J Pediatr Otorhinolaryngol ; 69(9): 1269-74, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15955571

ABSTRACT

Aero-digestive foreign bodies are a common occurrence in infants and children. The manifestations, hazards and consequences depend upon the location, nature of the foreign body and the time lapse. This communication reports a series of four cases with unusual course and consequences of aero-digestive foreign bodies. These were generalized tetanus, hypernatremia and metabolic acidosis with intra-cranial hemorrhage and severe pneumonia and empyema. Awareness of these consequences would help treating doctors to take appropriate steps in devising preventive and therapeutic strategies.


Subject(s)
Foreign Bodies/complications , Respiratory System , Upper Gastrointestinal Tract , Acidosis/etiology , Bronchi , Bronchiectasis/etiology , Bronchoscopy , Child , Child, Preschool , Esophagoscopy , Fatal Outcome , Female , Foreign Bodies/diagnosis , Humans , Hypernatremia/etiology , Infant , Intracranial Hemorrhages/etiology , Male , Nasal Cavity , Pneumonia, Aspiration/etiology , Tetanus/etiology
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