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VIRAL VECTORS. Abstract. For many human pathogens the traditional vaccine development platforms are unsuitable Owing to safety concerns Poor efficacy & Impracticality The alternative is therefore recombinant viral vectors as a means of vaccination

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Abstract
Abstract

  • For many human pathogens the traditional vaccine development platforms are unsuitable

  • Owing to safety concerns

  • Poor efficacy &

  • Impracticality

  • The alternative is therefore recombinant viral vectors as a means of vaccination

  • Can express foreign protein at high levels in host cells

  • Resulting in strong, long lasting immune responses


Introduction
Introduction

  • As compared to killed or inactivated virus vaccines, live attenuated vaccines produce better results

  • However for some human pathogens safety issues arise due to under attenuation and even reversion to its pathogenic state

  • e.g HIV

  • Live attenuated simian immunodeficiency virus (SIV)

  • Un fortunately SIV can also cause AIDS in monkeys due to reversion to pathogenecity


  • Even licensed live attenuated vaccines are not without the risk of vaccine mediated disease

  • Small pox vaccination

  • Vaccinia virus was used

  • This has significant side effects that can be even life threatening

  • Some viruses e.g Ebola and Marburg viruses are so deadly that live attenuated vaccine is not even considered

  • Even subunit and recombinant vaccine might contain post translational modifications after expression in host cells that might alter their antigenicity

  • These can also be degraded by the recipient immune system


  • In case of DNA vaccines all the discussed problems are eliminated

    But

  • Expression of DNA vaccines is notoriously weak in vivo, resulting in lower immunogenecity of the vaccine

  • Most promising recombinant vaccine technology platform is viral vectors

  • Idea is to present the naturally occurring forms of the target pathogen’s antigens to the immune system in the absence of the pathogen itself

  • Similar to natural infection however in the absence of the disease

  • 1984, vaccinia virus vector to express rabies virus glycoprotein (Raboral VR-G)


Most studied viral vectors as vaccine tools eliminated

  • Adeno virus

  • Adeno-associated viruses

  • Alphaviruses

  • Newcastle disease virus

  • Poxviruses

  • Vesicular stomatitis virus, etc


Adenovirus
ADENOVIRUS eliminated

  • Most widely studied viral vector for vaccine and gene therapy

  • Family: Adenoviridae

  • Double stranded DNA viruses with genome of approx: 36kbp

  • Of the human adenoviruses there are at least 51 different serotypes

  • Ad 2 and Ad 5 are most well studied in terms of viral vectors

  • Genomes are easy to manipulate, can be grown and purified to high titers in cell cultures and are able to infect a wide variety of dividing and non dividing cell types

  • Also have a favorable safety profile


ADENOVIRUS GENOME eliminated


  • It has been established that Ad genome can package upto 1.8kb of exogenous DNA (transgene)

  • Later it was found that additional space for transgene can be created by specific deletion of Ad genome

  • The first of these deleted genes were E1a and E1b which produce a replication incompetent vector due to loss of E1

  • This replication incompetent vector can only replicate in permissive cell line HEK293 which provides the missing E1 gene function for Ad vector

  • Additional deletions are made in E3 gene

  • These E1/E3 deleted Ad vector provide 4-5kb space for transgenes (first generation Ad vectors)

  • Efforts for low expression of Ad genes and high expression of gene inserts were made


  • This was achieved through deletions in the E2 region or the E4 region which reduced the expression of E2 and E4 genes

  • These Ad vectors are referred to as second generation Ad vectors

  • With increased transgene capacity of upto 6-7kb

  • Ad vectors for the highest capacity for exogenous DNA are called “gutless” vectors

  • Consist of solely the exogenous DNA flanked by Ad inverted terminal repeat (ITR) and the Ad packaging signal

  • These vectors can accommodate 30-35kb of foreign DNA

  • Although they must be propagated with a helper virus to provide the missing Ad genes necessary for replication in the packaging cell line


Summary of avv capacity
SUMMARY OF AVV CAPACITY E4 region which reduced the expression of E2 and E4 genes


Ad vectors have been studied on many fronts E4 region which reduced the expression of E2 and E4 genes

  • Ebola

  • Dengue

  • Marburg

  • Avian influenza

  • West Nile virus

  • SARS-CoV

  • HIV and

  • Anthrax

  • Also have been studied as gene therapy vectors for various cancers


  • Primary criticism is the issue of pre-existing immunity E4 region which reduced the expression of E2 and E4 genes

  • 35-55% of the Ad population has neutralizing Abs, in particular against Ad5

  • These Abs might limit Ad based vaccines vector’s efficacy

  • Use of alternate serotypes that are antigenically different and thus cannot be neutralized by these Abs is an option in this respect

  • However still conflicting data exists in this regard

  • Incase of Merck Ad-5 based HIV vaccine, the effect of neutralizing Abs can be overcome by increasing the dose of the vaccine in clinical trials



Adeno associated virus avv
Adeno-Associated Virus (AVV) (oral or intranasal) instead of injection can overcome pre-existing vector immunity

Belong to family Parvoviridae

Small single stranded DNA viruses with genome of ~ 5kb

8 known AVV serotypes, AAV 2 being the most commonly studied

Unique in sense that they need a helper virus to replicate e.g Ad virus or Herpes virus

In the absence of helper virus, infection becomes latent = no viral progeny

Wild type AVV do not produce disease in humans

Thus have excellent safety profile


Advantages (oral or intranasal) instead of injection can overcome pre-existing vector immunity

Broad host range

Persistent transgene expression in host cells

Generate very weak antivector immune responses

Recombinant AVV have been studied as vaccine vectors against Herpes simplex virus 2

HPV

HIV

Cytomegalovirus


rAVV can only accommodate ~5kb of exogenous DNA (oral or intranasal) instead of injection can overcome pre-existing vector immunity

This is done by deleting almost all of the vector genes between the 3’ and 5’ terminal repeat sequences

Must be packaged in special cell lines that express AVV rep and Cap proteins

In addition to a helper virus

Advantage is that there is no expression of parent virus genes, resulting in low antivector immunity

Disadvantage is that in absence of helper virus the viral genome gets integrated into the human genome at chromosome 19


Thus raises safety issues (oral or intranasal) instead of injection can overcome pre-existing vector immunity

Related to genetic consequence of genome integration (both beneficial and detrimental)

Pre-existing Immunity

Over 90% of humans have circulating Abs that cross react with AVV

And 30% are serotype positive for AVV neutralizing Abs

Highest level of AVV Abs are against AVV2 serotype

Alternate serotypes as vector backbone can be used

On July 26th 2007 U.S FDA announced the death of a clinical trial participant involving an AVV based RA treatment

Details of the tragedy have still not been released


Alphavirus
AlphaVirus (oral or intranasal) instead of injection can overcome pre-existing vector immunity

Belong to family Togaviridae

Small enveloped viruses, with single stranded positive RNA genome of ~ 11.8kb

Arthropod borne viruses(arbovirus) and are grouped into 6 clades (based on antigenic homology of E1 glycoprotein)

Barmah Forest (BF)

Ndumu (NDU)

Semliki Forest (SF)

Western equine encephalitis (WEE)

Eastern equine encephalitis (EEE)

Venezuelan equine encephalitis (VEE)


Have a broad host range and can infect a variety of cell types including dentritic cells (APCs) (can directly target DCs and produce strong immune responses)

Primary method of using alphaviruses as vaccine vectors is to create REPLICONS

By deleting structural genes and replacing them with transgenes

The recombinant RNA must be co expressed in packaging cell line with helper RNA containing the missing structural genes

Thus the replicons are enveloped viral particles containing the recombinant genome that when expressed in the cell line produces the transgene at high levels

One pitfall is the recombination between helper RNA and recombinant RNA

Which produces replication competent alphavirus particles


The genome capacity of alphaviruses is also low types including dentritic cells (APCs) (can directly target DCs and produce strong immune responses)

~5kb

These have been studied as vaccine vectors for avian influenza

Marburg virus

Ebola virus

HIV

SARS-CoV

Anthrax &

Botulinum toxin


VEE replicons containing HIV genes were tested in phase 1 clinical trials and were well tolerated in vaccine recipients

Pre-existing Immunity

Not as significant as in the case of Ad viruses and AAV

Largely because these are zoonotic , mosquito borne viruses that are endemic only in some geographical regions of the world

Also human alphavirus epidemics occur very in frequently

However there is evidence that pre existing antibodies in horses against one alphavirus strain can interfere with infection of another strain


New castle disease virus ndv
New Castle Disease Virus (NDV) clinical trials and were well tolerated in vaccine recipients

Belongs to family Paramyxoviridae

Zoonotic virus that infects all species of birds

Nonsegmented, single stranded negative sense RNA of ~15kb

Antigenically different from any of human paramyxoviruses

Categorized into 3 groups

The avirulent lentogenic strains

Moderately pathogenic mesogenic strains

Highly pathogenic velogenic strains


Lentogenic strains are widely used for NDV live attenuated vaccines in the poultry industry

NDV are nonpathogenic in primates and thus has lead to their study as vaccine vectors

Have been studied against SARS-CoV

Respiratory syncytial virus

SIV

Influenza virus

Despite the danger of NDV in poultry, these viruses have a safe profile in humans

Since NDV is an avian paramyxovirus, the issue of pre existing immunity is not considered

Also are used in many veterinary vaccines and are well characterized


NDV vectors may be somewhat limited in their capacity for large transgene inserts

Most recently Sendai virus vectors have been shown to accommodate 4.5 kb of exogenous DNA


Pox viruses
Pox Viruses large transgene inserts

Belong to family Poxviridae

Large double stranded DNA viruses

Viron size 350 x 270nm

Genome size 300kb

Most virulent virus is the variola virus an obligate human pathogen that causes smallpox

Other noteable viruses are

Vaccinia virus

Monkeypox virus

Cowpox virus (zoonotic viruses)


Poxviruses of the genus large transgene insertsAvipoxvirus have attained research attention as vaccine candidates

Are zoonotic arboviruses that are nonpathogenic in humans

Also have genome size of 260kb

Fowlpox and canarypox have been tested in animal models as vaccine vectors for rabies

H5N1 avian influenza

Nipah virus

HIV

Since these are zoonotic viruses, pre existing immunity is not considered to be an issue

Vaccinia viruses have been used as vaccines for decades


In case of vaccinia virus, much of the adult human population today is seropositive for vaccinia due to childhood smallpox vaccination

Therefore a vaccinia based vaccine vector in these individuals would be in effective due to antivector immunity

The large genome size of poxvirus is both advantageous and disadvantageous

Large size can accommodate large transgenes, but

The expression of parent virus proteins can produce strong immune responses that lead to reduced vaccine efficacy

This could be an explanation of poor performance of poxviruses in human clinical trials

In direct comparison a recombinant Ad vector expressing a transgene induces much stronger cellular immune responses than a vaccinia virus expressing the same gene


Vesicular stomatitus virus vsv
Vesicular Stomatitus Virus (VSV) population today is seropositive for vaccinia due to childhood smallpox vaccination

Belong to Rhabdoviridae (same family as rabies virus)

Zoonotic arboviruses

11kb genome of single stranded negative sense RNA

VSV transmission to animals takes place through insect bites

Can cause severe disease in cattle, horses and swine with symptoms similar to foot and mouth disease

In humans infections occur less frequently and also with less severe disease

In form of mild flu like symptoms

rVSV can accommodate a 40% increase in genome size with only a slight reduction in infectivity titer


Another advantage is that the virus can efficiently incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles

Concerns for VSV vector safety are related to severe human disease

As well as neurovirulence and 50% mortility rate from experimental intranasal mouse infections

Asymptomatic brain infections have also been noted in experimental models after intranasal delivery of VSV vector


rVSV vaccine vectors have still been studied against H5N1 incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles

Ebola and

Marburg viruses

Plague

Hepatitis C virus

HIV

Pre existing immunity is not considered an issue in this case as well

However in cases where it does exist, the option of serotype rotation is open, similar to Ad and AAV vaccine vectors


Other viruses
Other Viruses incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles

Herpes simplex virus (SIV, HIV and bacterial pathogens)

Measles virus (HBV, HIV and West Nile Virus)

Poliovirus (HBV and SIV)


Summary
Summary incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles

Viral vectors are suitable for presenting naturally formed antigens to the immune system

Have more favorable safety profile than live attenuated vaccines

Are more immunogenic than inactivated or killed virus vaccines

Present the desired antigens in the correct conformation in comparison to subunit vaccines

Express high levels of foreign genes in vivo than DNA vaccines


Considerations incorporate and express foreign transmembrane proteins on the surface of recombinant viral particles

Safety (Ad vectors considered generally safe but VSV are still in infancy stage and human safety is yet to be tested)

Pre existing immunity (for Ad and vaccinia virus it is serious while for zoonotic vaccine vectors it is not problematic)

Vector’s genomic capacity for a transgene insert (exogenous DNA can be in the range of 1 kb to 35kb depending on the vector)


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