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Towards a Coronavirus-Based HIV Multigene Vaccine

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Human immunodeficiency virus (HIV) infection represents one of the major health threats in the developing world. The costly treatment of infected individuals with multiple highly efficient anti-HIV drugs is only affordable in industrialized countries. Thus, an efficient vaccination strategy is required to prevent the further spread of the infection. The molecular biology of coronaviruses and particular features of the human coronavirus 229E (HCoV 229E) indicate that HCoV 229E-based vaccine vectors can become a new class of highly efficient vaccines. First, the receptor of HCoV 229E, human aminopeptidase N (hAPN or CD13) is expressed mainly on human dendritic cells (DCs) and macrophages indicating that targeting of HCoV 229E-based vectors to professional antigen presenting cells can be achieved by receptor-mediated transduction. Second, HCoV 229E structural genes can be replaced by multiple transcriptional units encoding various antigens. These virus-like particles (VLPs) containing HCoV 229E-based vector RNA have the ability to transduce human DCs and to mediate heterologous gene expression in these cells. Finally, coronavirus infections are associated with mainly respiratory and enteric diseases, and natural transmission of coronaviruses occurs via mucosal surfaces. In humans, HCoV 229E causes common cold by infecting the upper respiratory tract. HCoV 229E infections are mainly encountered in children and re-infection occurs frequently in adults. It is thus most likely that pre-existing immunity against HCoV 229E will not significantly impact on the vaccination efficiency if HCoV 229E-based vectors are used in humans.
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Towards a coronavirus-based HIV multigene vaccine
KLARA K. ERIKSSON, DIVINE MAKIA, REINHARD MAIER, BURKHARD LUDEWIG, &
VOLKER THIEL
Research Department, Kantonal Hospital Saint Gallen, Saint Gallen 9007, Switzerland
Abstract
Human immunodeficiency virus (HIV) infection represents one of the major health threats in the developing world. The costly
treatment of infected individuals with multiple highly efficient anti-HIV drugs is only affordable in industrialized countries.
Thus, an efficient vaccination strategy is required to prevent the further spread of the infection. The molecular biology of
coronaviruses and particular features of the human coronavirus 229E (HCoV 229E) indicate that HCoV 229E-based vaccine
vectors can become a new class of highly efficient vaccines. First, the receptor of HCoV 229E, human aminopeptidase N
(hAPN or CD13) is expressed mainly on human dendritic cells (DCs) and macrophages indicating that targeting of HCoV
229E-based vectors to professional antigen presenting cells can be achieved by receptor-mediated transduction. Second,
HCoV 229E structural genes can be replaced by multiple transcriptional units encoding various antigens. These virus-like
particles (VLPs) containing HCoV 229E-based vector RNA have the ability to transduce human DCs and to mediate
heterologous gene expression in these cells. Finally, coronavirus infections are associated with mainly respiratory and enteric
diseases, and natural transmission of coronaviruses occurs via mucosal surfaces. In humans, HCoV 229E causes common cold
by infecting the upper respiratory tract. HCoV 229E infections are mainly encountered in children and re-infection occurs
frequently in adults. It is thus most likely that pre-existing immunity against HCoV 229E will not significantly impact on the
vaccination efficiency if HCoV 229E-based vectors are used in humans.
Keywords: AIDS, vaccination, coronavirus, HIV
Abbreviations: HIV, human immunodeficiency virus; DCs, dendritic cells; AIDS, acquired immunodeficiency syndrome;
HCoV, human coronavirus; MHV, mouse hepatitis virus
Introduction
Prophylactic vaccines against several viral infections
have been developed over the last centuries leading to
the eradication of smallpox and protecting many
people from diseases such as measles, rubella, mumps
and polio. However, a number of diseases remain
against which current vaccines are suboptimal or
unavailable. Furthermore, there is growing need to
develop therapeutic vaccines which may boost specific
immune response to persistent viruses such as human
immunodeficiency virus (HIV). The critical first step
in the development of antiviral vaccines is the
identification of the dominant antigens contributing
to the different stages of the infection, i.e. initial
replication at the site of entry, spread in the host and
establishment of a persistent infection. The metho-
dology for the identification of antigens and the
characterization of immunodominant epitopes is well-
established and has been further advanced by
approaches from the elds of proteomics and
genomics (Chakravarti et al. 2000). However, the
major bottle-neck in the development of new and
effective vaccines is the delivery of antigens to cellular
components of the immune system that initiate
protective antiviral immunity. The unmatched
capacity of dendritic cells (DCs) to sample antigen
at sites of pathogen entry, transport pathogens and
their immunogenic components to secondary
ISSN 1740-2522 print/ISSN 1740-2530 online q 2006 Taylor & Francis
DOI: 10.1080/17402520600579168
Correspondence: V. Thiel, Research Department, Kantonal Hospital Saint Gallen, Saint Gallen 9007, Switzerland. Tel: 41 71 494 2843.
Fax: 41 71 494 6321. E-mail: volker.thiel@kssg.ch
Clinical & Developmental Immunology, June December 2006; 13(24): 353–360
lymphoid organs and to initiate activation of T cells
make them the ideal target cell for antimicrobial
vaccines (Steinman and Pope 2002). The excellent
capacity of DCs to prime antiviral T cell responses can
be readily shown in vitro, i.e. few DCs can activate
large numbers of virus-specific T cells in a mixed
lymphocyte culture (Macatonia et al. 1989; Nonacs
et al. 1992). An important prerequisite for the
initiation of immune responses in vivo is the
translocation of antigens from peripheral sites into
secondary lymphoid organs (Zinkernagel et al. 1997).
The high potency of DCs to induce protective antiviral
immunity against the non-cytopathic lymphocytic
choriomeningitis virus (LCMV) in vivo has been
shown in studies where only 100 1000 DCs present-
ing a specific viral antigen have to reach secondary
lymphoid organs for the induction of protective
antiviral T cell responses (Ludewig et al. 1998;
Ludewig et al. 2000b). DC-induced CTL responses
develop rapidly and DC-immunized mice are pro-
tected against acute systemic and peripheral viral
challenge (Ludewig et al. 1999). Likewise, adoptive
transfer of DCs pulsed with inactivated HIV-1 into
severely immunocompromised mice reconstituted
with human PBL resulted in the induction of
protective anti-HIV-1 responses (Lapenta et al.
2003). Moreover, vaccination of SIV-infected rhesus
monkeys with a cellular DC vaccine significantly
suppressed viral replication (Lu et al. 2003). A recent
study in untreated HIV-1 infected individuals revealed
that DC-based vaccination can elicit potent immune
responses against immunodeficiency viruses in
humans (Lu et al. 2004).
The efficacy of vaccines can be enhanced if optimal
activation/maturation of DCs is achieved. For
example, DC maturation via toll-like receptor ligands
augments the activation of cytomegalovirus- and HIV-
specific T cell responses in vitro (Lore et al. 2003).
Likewise, the efficiency of various vaccine formats can
be greatly enhanced if activation of DCs in vivo is
mediated via co-delivery of immunostimulatory
oligonucleotides (Sparwasser et al. 1998; Ludewig
et al. 2000a) or binding to heat shock proteins
(Cho et al. 2000). Incorporation of DC-activating
chemokines or factors prolonging DC survival into
genetic vaccines has been shown to enhance the
immune response against recombinant rabies virus
(Pinto et al. 2003) or HIV gp120 (Biragyn et al. 2002).
A potent and effective HIV vaccine should thus
directly deliver antigens to DCs and induce their
activation/maturation.
HIV infection and immunity
Prevention of HIV infection. The thorough knowledge of
the biology of HIV that has been generated over the last
two decades has paved the way for a rational vaccine
design. Furthermore, the progress in the understanding
of the basic immunological mechanisms underlying
antigen presentation (Steinman and Pope 2002),
lymphocyte trafficking and activation (Luther and
Cyster 2001), and immunological memory (Kaech
et al. 2002) has been instrumental for the identification
of the relevant parameters that ensure the induction
of protective antiviral immunity. Accordingly, an
efficient HIV vaccine should induce long-lasting,
broad humoral and cellular responses against the
immunodominant HIV antigens. In particular, the
vaccine should (i) target and activate DCs, (ii) contain
the immunodominant antigens recognized by CTL and
Th cells, (iii) be able to display antigenic determinants
that induce broadly neutralizing antibody responses,
and (iv) be applicable via mucosal surfaces.
HIV-specific CTL and Th cell responses. CTL responses
crucially contribute to control of immunodeficiency
virus infection. Broad virus-specific CTL responses can
be found in peripheral blood of HIV-infected humans
(Betts et al. 2001; Addo et al. 2003) and the decline of
plasma viral RNA during primary HIV infection is
associated with the appearance of HIV-specific CTL
(Borrow et al. 1994; Koup et al. 1994). Furthermore,
transient in vivo depletion of CD8 T cells lead to a
massive increase in viral load in SIV-infected monkeys,
whereas extension of the depletion for more than 28
days elicited a progressive AIDS-like syndrome(Jin et al.
1999; Schmitz et al. 1999). HIV-specific Th cells can be
detected in infected individuals (Pitcher et al. 1999). It
is, however, not yet clear whether these cells extert direct
antiviral effects. However, the good correlation of
functional CD4 T cell responses against HIV
(Rosenberg et al. 1997) or SIV (McKay et al. 2003)
with the clinical status strongly supports the notion that
intact Th cell responses are instrumental for long-term
virus control. This is most likely mediated indirectly by
stimulation of virus-specific CTL. Since most patients
develop T cell responses against the HIV proteins env,
gag or nef (Betts et al. 2001; Addo et al. 2003), a broadly
applicable vaccine should elicit immune responses
(at least) against these three immmunodominant
antigens.
Broadly neutralizing antibodies. Non-neutralizing
antibodies directed against viral proteins appear early
after HIV infection, whereas neutralizing antibodies
appear usually rather late after primary infection
(Pilgrim et al. 1997). Furthermore, sera from HIV-
infected individuals usually display only weak
neutralizing activity against primary isolates (Moore
et al. 1995). The fact that depletion of B cells in
Rhesus monkeys significantly delayed the appearance
of neutralizing antibodies but did not impact on the
early viral clearance (Schmitz et al. 2003) supports the
notion that neutralizing antibodies do not contribute
K. K. Eriksson et al.354
significantly during initial HIV infection. However,
the presence of neutralizing antibodies may alter the
clinical course of SHIV infection in macaques and
prevents peripartal infection (Baba et al. 2000).
Conventional vaccination approaches consistently
failed to induce broadly neutralizing antibody
responses (McMichael and Hanke 2003).
Nevertheless, distinct monoclonal antibodies have
been described that are capable of neutralizing a
broad range of different HIV isolates, suggesting that
such antibody responses might be induced once an
adequate vaccination strategy has been developed
(Moore et al. 2001). For example, altering the
immunodominance pattern by using CD4-HIV
envelope fusion constructs that expose normally
occluded and conserved antigenic regions represents
such an approach for the induction of broadly
neutralizing antibodies (Fouts et al. 2003).
An alternative strategy for the induction of antibodies
that inhibit the infection of primary T cells with
different primary HIV-1 isolates has been reported
recently. This promising approach takes advantage of
the highly conserved caveolin-1 binding domain of
HIV-1 glycoprotein 41. Neutralization of the
caveolin-1 binding site in gp41 efficiently blocks
HIV-1 entry in a wide range of primary cells
(Hovanessian et al. 2004).
Mucosal vaccination. HIV is predominantly transmitted
via mucosal surfaces (Pope and Haase 2003). For
example, SIV rapidly crosses the epithelial layers in the
cervical mucosa and infects predominantly DCs and
CD4 T cells (Spira et al. 1996). Following primary
infection, the virus gains access to lymphoid organs and
establishes persistent infection in CD4 T cells and
macrophages. It appears that constant low-level
exposure to virus (via mucosal surfaces?) is associated
with resistance to HIV infection (Zhu et al. 2003).
Mucosal vaccination may block transmission of
intravaginally or intrarectally applied SIV (Amara
et al. 2001; Belyakov et al. 2001; Veazey et al. 2003)
indicating that an HIV vaccine should prevent the early
stage of infection and elicit long-lasting mucosal
immunity.
Coronavirus biology and suitability as viral
vectors
Although immunogenic peptides or naked nucleic acid
can elicit immune responses against HIV antigens, the
use of viral vectors represents a superior strategy to
deliver HIV antigens and/or immunostimulatory cyto-
kines to specific target cells. However for several
reasons, many virus vector systems are still limited in
their ability to induce a broad and long-lasting antiviral
immune response capable to prevent HIV infection
and/or to reduce viral load. Moreover, the safety of
DNA-based vectors such as adeno-associated-, retro- or
lenti-viruses is a matter of concern, because they can
integrate into the host cell genome (Dobbelstein
2003). Recombinant adenoviruses have been studied
intensively as HIV vaccine candidates mainly because
they can be produced to high titers. Nevertheless, high
doses of recombinant adenovirus vectors have to be
applied to induce antiviral immune response, most
probably because they target antigens mainly to non-
lymphoid organs such as the liver (Krebs et al. 2005).
In contrast to viral vectors based on DNA viruses, the
use of positive-stranded RNA virus-based vectors
that replicate in the cytoplasm are considered as safe
vectors because it is unlikely that sequences from these
vectors can integrate into the host cell genome.
Moreover, the safety is well documented for vectors
based on widely used vaccine strains such as poliovirus
(Crotty et al. 1999) or virus-like particles (VLPs) that
contain replicon RNAs devoid of structural genes
(Davis et al. 2000; Harvey et al. 2003). Although
some of these vectors are able to target DCs and/or
to induce mucosal immunity, their cloning capacity
is generally restricted and the expression of multiple
HIV antigens and/or immunostimulatory cytokines is
limited.
Coronaviruses display a number of features that
may be advantageous to overcome these limitations
and, therefore, represent promising candidate vaccine
vectors. Coronaviruses are enveloped viruses that are
associated mainly with respiratory and enteric
diseases. For example, human coronavirus 229E
infects the mucosa of the upper respiratory tract and
can cause common cold. Coronavirus genomes are the
largest known autonomously replicating RNAs with a
size of approximately 30 kb. About two thirds of the
positive-stranded genome encode the replicase gene,
which is comprised of two large open reading frames
(ORFs). Upon infection, translation of the genomic
RNA results in the synthesis of replicase gene-encoded
polyproteins that are extensively processed by viral
proteinases leading to the formation of a functional
replicase transcriptase complex within the cytoplasm
of the infected cell (Ziebuhr et al. 2000). A hallmark of
coronavirus genome expression is their unique
transcription strategy. This strategy leads to the
synthesis of multiple 3
0
co-terminal subgenomic
mRNAs, encoding mainly structural proteins. It has
been shown that the synthesis of each subgenomic
mRNA involves a discontinuous step by which the so-
called 3
0
body sequence is fused to the genomic 5
0
leader sequence (Spaan et al. 1983). The fusion of
leader and body sequences during discontinuous
transcription is determined, at least in part, by cis-
acting elements, termed transcription regulatory
sequences (TRS, also referred as transcription
associated sequences). These elements are located at
the 5
0
end of the genome and at various 3
0
proximal
sites corresponding to the individual transcription
A coronavirus-based HIV multigene vaccine 355
units. Although many studies have been performed to
identify cis-acting sequences required for coronavirus
transcription, exact borders of TRS elements have not
yet been elucidated (Pasternak et al. 2001). However,
short stretches of not more than 5 7 nucleotides
within the TRS, called “core sequence”, have been
identified to determine the site of leader-body fusion
of coronavirus subgenomic RNAs.
Because of the (molecular) biology of coronaviruses,
coronavirus-based vectors are currently considered
a promising system to genetically deliver multiple
heterologous genes to specific target cells. First,
coronaviruses are positive-stranded RNA viruses
replicating in the cytoplasm without a DNA inter-
mediary, making insertion of viral sequences into the
host cell genome unlikely. Second, coronaviruses have
the largest RNA genome known so far. Therefore, a
cloning capacity of more than 6 kb is expected. Third,
coronaviruses display a unique transcription process
resulting in the synthesis of 68 subgenomic mRNAs,
encoding mainly the structural genes. These genes,
encoded at the 3
0
third of the genome, can be replaced
by multiple heterologous genes, e.g. immunogenic HIV
antigens and/or immunomodulatory genes. Fourth, the
receptors of human and murine coronaviruses (HCoV
229E and mouse hapatitis virus (MHV)) are expressed
on human and murine DCs, respectively, indicating
that efficient delivery (i.e. receptor-mediated uptake of
VLPs) of heterologous genes to DCs can be achieved.
Finally, the mucosal route is the natural way of
coronavirus transmission.
Establishment of a reverse genetic system for
coronaviruses
We have established a reverse genetic system for
coronaviruses that allows the generation of recombinant
coronaviruses (Thiel et al. 2001a, 2003; Coley et al.
2005). One of the main advantages of our system is that
the cloned full-length coronavirus cDNAs are amenable
to site-directed mutagenesis using vaccinia virus-
mediated homologous recombination. This technique
is well established and has been proven to represent
an efficient and precise (on the nucleotide level) method
to genetically modify recombinant coronavirus cDNAs.
In Figure 1, we show one example to demonstrate
the ease of using vaccinia virus-mediated recombination
to genetically modify coronavirus cDNA inserts.
Generation of coronavir us-based multigene vector
RNAs—transduction of human DCs. With the reverse
genetic systems available, it is now possible to make
use of the unique characteristics of coronavirus
transcription to develop coronavirus expression
vectors. The rationale of expressing heterologous
genes using coronavirus-mediated transcription is to
insert a transcriptional cassette, comprised of a
coronavirus TRS located upstream of the gene of
interest, into a coronavirus genome, minigenome or
vector RNA. We have shown for human coronavirus
vector RNAs that a region of at least 5.7 kb is
dispensable for discontinuous transcription (Thiel
et al. 2001b). This region contained all structural
genes and, therefore, our vector RNAs are not
infectious. We could demonstrate that it is possible
to construct a human coronavirus vector RNA capable
to mediate the expression of multiple heterologous
proteins. Noteworthy, this vector RNA can be
packaged to VLPs if the structural proteins are
expressed in trans (Thiel et al. 2003). These results
indicate that coronavirus-based vector systems might
be useful for heterologous gene expression, especially
for longer and multiple genes.
Figure 1. Mutagenesis of cloned coronavirus cDNA using vaccinia virus-mediated homologous recombination. (A) The generation of the
recombinant vaccinia virus vVec-GN containing a HCoV 229E-based vector construct is illustrated. Two steps of recombination using the E.
coli guanine phosphosribosyltransferase (gpt) as marker for positive and negative selection were performed. (B) The result of a PCR analysis
from vaccinia viruses vHCoV-inf-1 (parental clone), vRec-1 (intermediate clone) and vVec-GN (desired final clone) is shown. PCR primers
used in this analysis are located upstream and downstream of the region where recombination took place. Lanes 1 12 show 12 randomly
picked recombinant vaccinia virus plaques obtained after gpt-negative selection, indicating the 100% recovery of desired genotypes. Notably,
one vVec-GN clone was subjected to sequencing analysis of the entire coronavirus-based cDNA insert and no nucleotide changes were
detected.
K. K. Eriksson et al.356
An important consideration for viral vaccine vectors
is their potential for efficient delivery of their genetic
material to specific target cells. For example, targeting
of viral vaccine vectors to DCs is highly desirable in
order to optimize vaccine efficacy. It is important to
note that the HCoV 229E receptor, human amino-
peptidase N (hAPN or CD13), is expressed at high
levels on human DCs (Summers et al. 2001). This
implies that HCoV 229E-based VLPs could be used to
efficiently (receptor-mediated uptake) transduce these
cells. We could demonstrate that HCoV 229E-based
VLPs can be used to transduce immature and mature
human DCs (Thiel et al. 2003). Therefore, this new
class of safe, multigene vectors, based on HCoV 229E,
represents a particularly promising tool to genetically
deliver multiple antigens and immunostimulatory
cytokines to human DCs.
A reverse genetic system for mouse hepatitis
virus (MHV)—establishment of a murine model
to assess the efficacy of coronavirus-based
vaccine vectors
In order to study the efficacy of coronavirus-based
vectors in vivo, a small animal model is desirable.
Therefore, we first established a reverse genetic system
for MHV. Again we made use of vaccinia virus as
cloning vector to stably propagate the full-length cDNA
of MHV (strain A59). Recombinant viruses obtained
from this cDNA clone were indistinguishable from the
parental MHV-A59 strain in tissue culture (growth
kinetics, plaque size and RNA synthesis) and in MHV-
related disease models in mice (Coley et al. 2005).
With the reverse genetic system for MHV it is now
possible to generate MHV-based multigene vectors
that resemble their HCoV-229E counterparts. Like all
coronaviruses, MHV mediates the expression of
multiple subgenomic mRNAs in the infected cell.
Therefore, it is possible to use the coronavirus
transcription mechanism for the generation of multi-
gene MHV vectors. Furthermore, MHV is one of the
best-studied coronaviruses in vitro and in vivo. MHV
grows to high titers in tissue culture (. 10
9
pfu/ml)
and the requirements for the generation of VLPs are
well understood. MHV also allows for the usage of a
collection of well characterized inbred and transgenic
mice and a variety of established immunological
techniques, indispensable for the analysis of vector-
induced immune responses. Finally, it has been shown
that MHV-A59 can infect murine DCs (Turner et al.
2004) and therefore, recombinant MHV vectors in the
context of a murine model can serve as a paradigm for
the development and evaluation of coronavirus
vaccine vectors.
An important prerequisite to study the efficacy of
coronavirus vaccine vectors is the availability of VLPs
that can be produced to high titers. Therefore,
packaging cell lines must be established which mediate
the expression of coronavirus structural proteins in
trans. To this end, we have generated several cell clones
derived from murine 17-clone1 cells, which stably
express the MHV structural proteins E and M
(EM-cells). These clones have been analysed for the
expression of E and M by PCR using genomic DNA as
template and RT PCR using poly(A)-containing
RNA as template (Figure 2A), and immunofluorescent
Figure 2. Packaging cell lines for the production of MHV VLPs. (A) Genomic DNA (left) or polyA-containing RNA (right) from cell clones
(#2,4,8,9,12 and13) that were stably transfected with a plasmid DNA encoding the MHV E and M genes on separate transcription units were
analyzed by E and M gene-specific PCR and RTPCR, respectively. Control lanes are genomic DNA or polyA-containing RNA from parental
murine 17-clone1 cells, plasmid DNA encoding E and M and polyA-containing RNA from MHV-infected 17-clone1 cells. (B)
Immunofluorescence analysis of 17-clone1-EM13 cells using specific sera against MHV structural proteins E (left) and M (right). (C)
Packaging strategy for the production of MHV VLPs using the MHV prototype vector MHV-Vec-A and the E- and M-expressing cell lines.
A coronavirus-based HIV multigene vaccine 357
microscopy (Figure 2B). Five out of six cell clones
were found to contain and express both, E and
M. Noteworthy, two considerations have been made
before the construction of the EM cell line. First, the
E and M genes in these cell lines are expressed by
the cellular transcription of two separate mRNAs to
minimize the possibility of reconstitution of infectious
viruses by recombination of the MHV vector RNA with
E and M gene mRNAs (Figure 2C). Second, in order to
achieve high titer MHV VLP production, we decided to
use a mouse cell line which is susceptible to MHV
infection (17-clone1) for the stable transfection of
MHV E and M genes. In this case, the packaging cells
are susceptible to VLP-infection and we expect spread
of MHV vector RNA throughout the tissue culture.
The E- and M-expressing cell lines can now be
used to package MHV vector RNAs that encode
(in addition to the replicase gene and the 5
0
and 3
0
cis-acting elements required for replication) the MHV
structural proteins S and N (nucleocapsid protein).
Therefore, we have generated a prototype MHV
vector, designated MHV-Vec-A, containing the repli-
case gene, the 5
0
and 3
0
cis-acting elements required for
replication, the structural protein S, the immunodo-
minant CTL epitope GP33 of LCMV glycoprotein as
a fusion protein with the green fluorescent protein
(GP33-GFP) and the nucleocapsid protein. This
vector RNA is currently being used to thoroughly
assess the efficacy of VLP production in individual
EM-packaging cells.
Our first experiments using MHV-Vec-A RNA for
the transfection of the packaging cell line clone “17-
clone1-EM13” showed that the transduction of these
cells yields green fluorescent plaques indicating that
our construct is functional, i.e. that the replicase
complex, and the GP33-GFP fusion protein are
produced. As expected, we could also observe syncytia
in vector-transfected packaging cells, suggesting that
a functional, cell fusion-mediating MHV S protein
is present (Figure 3, left panel). Most importantly,
the production of MHV VLPs is shown by the fact
that transfer of supernatants from vector-transfected
17-clone1-EM13 cells to primary DC cultures leads
to GFP expression in the target cells (Figure 3,
right panel). Overall, these experiments provide
proof-of-principle that the generation of MHV VLPs
is feasible and that transgenes expressed by these
replication-incompetent viruses can be targeted to
DCs. We are currently in the process of testing the
efficacies of MHV VLP production using the different
17-clone1-EM cell clones in order (i) to identify the
best packaging cell clone; and (ii) to establish an
optimized protocol for high titer VLP production.
Conclusions
The human immunodeficiency virus (HIV) pandemic
with approximately 40 million people infected world-
wide and more than 4 million deaths per year,
represents a major human health problem. The
majority of the infections occur in Africa and HIV-
induced AIDS is the leading cause of death among
adults aged 15 49 years in this region. Furthermore,
the numbers of infections in developing countries such
as India and China have been dramatically growing
over the recent years. Antiviral drug treatment has
increased life expectancy and quality in western
countries, but this expensive medication is usually
not accessible for infected individuals in developing
countries. There is thus an urgent need for an efficient
and affordable vaccine.
Figure 3. Generation of MHV VLPs and in vitro transduction of primary dendritic cells. MHV-Vec-A RNA was transfected into 17-clone1-
EM13 cells by electroporation. Green fluorescence was detectable after 24 h (left). Supernatants from 17-clone1-EM13 cells were collected
between 48 and 72 h following transfection and used to transduce primary DCs in vitro. Again, green fluorescence became apparent after 24 h
(right) indicating that functional MHV VLPs have been produced in the 17-clone1-EM13 packaging cell line and that these VLPs can be used
to transduce murine DCs.
K. K. Eriksson et al.358
We believe that coronaviruses have tremendous
capability as tools to deliver prophylactic and
therapeutic proteins to disease-relevant target cells in
human. In addition, this inherently safe vector system
offers the opportunity to deliver multiple proteins in
combination with immunostimulatory substances.
The primary goal of the outlined approach is the
establishment of the coronavirus vector system and its
validation in a small animal model. If this approach is
feasible and effective, we should commence with the
development of HCoV 229E replicon-based VLPs
encoding several HIV antigens (env, gag and nef) in
combination with immunostimulatory molecules. The
successfully established packaging strategy will be
adapted to the HCoV 229E system and should allow
production of recombinant HCoV 229E VLPs.
Alternatively, pseudotyped MHV-based VLPs dis-
playing a tropism for human DCs may be used for
further studies. Safety and efficacy of this vaccine
preparation should be tested in an adequate non-
human primate model.
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... De esta forma las teorías de conspiración pierden fuerza (11,19,20). El virus SARS-CoV-2 detrás de la pandemia en curso, deja claro que evolucionó a partir de cepas naturales (17), según un nuevo análisis genómico de comparación (11,19). ...
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Den wichtigsten Zelltyp in der Immunaktivierungskaskade stellen die dendritischen Zellen (DCs) dar, die darauf spezialisiert sind, prozessierte Peptide den CD4+ und CD8+ T-Zellen zu präsentieren, aber auch mikrobielle Glykolipide für NK-Zellen sichtbar zu machen und native Antigene den B-Zellen zu präsentieren. Daher stellt eine effiziente Belieferung dieser spezialisierten Immunzellen mit Antigenen eine besondere Herausforderung für die Vakzineentwicklung dar. Neben der spezifischen Antigenbeladung, sollten die DCs auch adäquaten Maturationsstimuli ausgesetzt werden, um die vollständige Aktivierungskapazität dieser Zellen zu erhalten. Coronavirus basierte Vektoren sind vielversprechende Werkzeuge, um spezifische Zielzellen mit Antigenen zu beliefern. Diese Viren sind die Grössten der bekannten, positivsträngigen RNA-Viren und stellen eine Klonierungskapazität von 6-9 kb zur Verfügung. Zudem ermöglicht ihre Transkriptionsstrategie die Expression verschiedener Antigene und immunstimulatorischer Zytokine. Die Viren zeigen den weiteren Vorteil, dass sie bevorzugt professionelle Antigen präsentierende Zellen, DCs und Makrophagen, infizieren. Daher war das Hauptziel dieser Arbeit die Entwicklung eines Coronavirus basierten Systems, das den Transport von Antigenstrukturen hin zu Antigen präsentierenden Zellen, bevorzugterweise DCs, in sekundären lymphatischen Organgen gewährleistet. Im Kapital 5.1 dieser Arbeit wird die grundlegende Methodologie für die Generierung von rekombinanter Coronavirus RNA beschrieben. Basierend auf dieser Methodik wurden erste Schritte unternommen, um ein auf dem Maushepatitisvirus (MHV) basierendes System zu etablieren, das die Produktion von Virus ähnlichen Partikels (VLPs) ermöglicht. In Kapitel 5.2 wird eine erste Serie von Experimenten beschrieben, die die Deletion der Strukturproteine E und M des MHV in den Vektorkonstrukten zum Ziel hatten. Eine Verpackungszellinie wurde etabliert, die eine Komplementierung der fehlenden Strukturproteine in trans ermöglichte. Mit Hilfe dieser Zellinie wurde die Produktion von replikationsdefizienten, aber propagierungskompetenten VLPs möglich. Es konnte gezeigt werden, dass MHV-basierte VLPs, die eine Antigenkassette mit GFP exprimieren, primäre DCs in vitro transduzieren können. Da aber eine Produktion von grösseren Mengen von VLPs in diesem Ansatz nicht möglich war, wurde ein Protokoll entwickelt, dass die Generierung von hohen Titern der MHV-basierten VLPs ermöglicht. In Kapital 6.1 wird die Konstruktion eines Satzes von MHV-basierten Vektoren beschrieben, bei denen eine Attenuierung durch die Deletion aller akzessorischen Gene und des Gens für das Strukturprotein E und die funktionelle Ablation des Nichtstrukturproteins nsp1 erreicht wurde. Induzierbare Verpackungszellinien, die das virale Protein E in trans bereitstellen, wurden generiert und grosse Mengen an VLPs konnten mit Hilfe dieser Verpackungszellinien hergestellt werden. Diese VLPs waren stabil über mindestens 12 Passagen und waren propagierungsdefizient in Wildtypzellen. Im Kapitel 6.2 wird das Design einer Serie von VLPs beschrieben, die verschiedene Kombinationen von Transgenen tragen. Das erste Paar dieser VLPs vermittelt die Expression eines Fusionsproteins bestehend aus dem immundominanten Epitop (gp33-KAVYNFATC) des Glykoproteins des Lymphozytären Choriomeningitis Virus (LCMV-GP) und dem grün fluoreszierenden Proteins (GFP), allein oder gemeinsam mit dem immunstimulatorischen Zytokine Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF). Der zweite Satz von Vektoren kodiert für ein humans CTL Epitop des Melan-A Proteins, das mit Hefeubiquitin und GFP fusioniert ist. Auch diese Antigenkassette wurde entweder allein oder zusammen mit GM-CSF verwendet. Effiziente Tranduktion von DCs und Makrophagen mit Vektor vermittelter Antigenexpression in vitro und in vivo konnte demonstriert werden. Einmalige Immunisierung mit Mengen von 104 – 105 pfu führte zu starker und langanhaltender, protektiver, antiviraler und antitumoraler Immunität. Zusammenfassend kann festgehalten werden, dass diese neu entwickelte Vakzineplattform eine Lieferung von Antigenen und immunstimulatorischen Zytokinen zum Wirkort einer erfolgreichen und protektiven Immunanwort ermöglicht.
Chapter
IntroductionNomenclatures and SIV phylogenetic classificationSIVs and SIV/HIV hybrid viruses (SHIV) used for induction of AIDS, vaccine, and drug studiesNHP models of human HIV transmissionPathogenesis of SIV and SHIVImmune control of SIV and SHIVAIDS vaccine modelsPre- and postexposure prophylaxisImmunotherapy with vaccinesSummaryReferences
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