Dengue animal models

MICA (P) 193/06/2005

Proceedings of the Second Regional Dengue Research Network

Meeting
Srisakul (Susie) Kliks Ph.D - Vaccine Safety Research Program, PDVI, University of California Office
of the President, Oakland, California, USA.

The Second Regional Dengue Research Network Meeting co-sponsored by the

Pediatric Dengue Vaccine Initiative (PDVI) and the Novartis Institute for Tropical
Diseases (NITD) was held in Singapore September 28-30, this year. The twintheme of the meeting, Strategies for Vaccines and Therapeutics for Dengue
and other Flaviviral Diseases brought together 330 participants from Asia region
and outside. Forty seven oral and 121 posters covering a broader range of topics
were presented.
PDVI and NITD: Partners in the support of sciences for Dengue treatment/vaccines

The inception of PDVI for Dengue vaccine and NITD for Dengue drug discovery
in 2003 has provided a vital stimulus to dengue research as reflected in this
meeting. In ways that neither could do alone, the partnership is more able to
address fundamental issues common to the development and application of anti
Dengue drugs and vaccines than ever before.
NITDs progress with its Dengue drug discovery program reflected in this meeting
with presentations on both antiviral and host targets to inhibit dengue, along
with the development of in vitro high through- put screening assays and animal
models for screening candidates. In addition, DengueInfo Database, a joint effort
between the NITD and the Genome Institute of Singapore (GIS) was launched
to bank all published Dengue genome sequences associated clinical information
(if available) and other useful information such as location and isolation date
will be included in the database. The database, equipped with various search
tools, is freely accessible for academic and commercial users and can be found
at http://dengueinfo.org.
Research Highlights

High quality research on key dengue issues was evident in this meeting. Analyzing
the phylogenic pattern of Dengue genetic sequence of all serotypes, Dr. Edward
Holmes presented evidence for strong purifying selection through the back and
forth replication in its divergent hosts as in mosquitoes and humans. At the
same time, the complex immunological selection also exerts influence on the
intra-serotype genetic diversity.
To understand the molecular basis of antibody-mediated Dengue neutralization,
Dr. Richard Kuhn showed different binding patterns of several Dengue monoclonal
antibodies to the virus, utilizing the cryo-electron microscopy image and the
atomic structure of both the Dengue Envelope protein and Fab portions of several
monoclonal antibodies.
A collaborative study between Thailands Mahidol-Siriraj Medical Faculty and
Frances Pasteur Institute was presented by Dr. A. Sakuntabhai on the genetic
polymorphism of a promoter region of the DC-SIGN gene. This gene codes for
a receptor on the surface of dendritic cells involved in Dengue virus entry. A

distinct genetic motif in the promoter of this genes appears to be a factor in the
propensity of a person to develop Dengue Hemorrhagic Fever (DHF) or Dengue
Shock Syndrome (DSS) upon Dengue infection. However, whether dendritic cells
represent the main target cells, or the DC-SIGN receptor constitutes the primary
Dengue receptor requires further investigation.
Interestingly, Dr. Pierre-Yves Lozach at the Pasteur Institute reported that the
interaction between mannosylated N-glycan of the Dengue Envelope protein
and the DC-SIGN receptor on dendritic cells triggers rapid internalization of the
viral particle. However, when the DC-SIGN internalization signal was disabled,
the attached virus continued to be internalized, indicating involvement of an
alternative receptor on the dendritic cells. Therefore, Dengue specific receptor(s)
both in mammalian and insect cells remains a pressing issue with this research
community as evidenced in several posters from Japan, Thailand, Taiwan and
France. Many have already harnessed Dengue pseudoviruses as a tool to search
for dengue specific receptors.
Dr. Jeremy Farrars presentation on the clinical hallmarks of Dengue fever
and DHF drove home our need to pay close attention to the clinical
manifestations during the course of the diseases and to ask relevant questions
related to pathogenesis, therapeutics and vaccines. It was pointed out that
the experience gained by physicians in developing countries treating tropical
diseases like Dengue fever and DHF should be put to good use by clinicians
in countries where these diseases have only recently emerged or started to
take on epidemic proportions.
Relevant to the viral therapeutics and vaccines, Dr. Michael Diamond of
Washington University presented data on the mechanism of protective antibodies
against West Nile virus and its implications toward therapeutic antibodies and
vaccines. The study focused on a West Nile specific monoclonal antibodies
(mAbs) to domain III of the Envelope protein that neutralize an in vitro viral
infection in Vero cells, blocks ADE in mouse macrophages, and correlates to
protection against death by West Nile virus infection in mice. The neutralization
mechanism of these mAbs does not appear to be due to prevention of viral
attachment on the cell surface, but rather, the blocking of the viral fusion in the
endosomal vesicle, following the internalization of the viral-antibody complexes
into the cells.
New at this meeting were several new posters reporting on clinical studies
from Vietnam, Thailand, Netherlands/Indonesia and Taiwan.
Many thanks to the Scientific Program Committee, Local Organizing Committee
and Prof. Ng Mah Lee who provided excellent coordination to make the meeting
a success.
All oral and poster abstracts and the presentation slides will be posted at
http://www.pdvi.org and http://dengueinfo.org.

T his issue of Dengue Digest focuses on animal models for Dengue and
their role in research and the development of drugs and vaccines.
The guest editor of this issue is Dr. Wouter Schul from the Dengue Unit at NITD.
Dr. Schul obtained his Ph.D. from the University of Amsterdam, the Netherlands,
was a postdoctoral fellow at the Erasmus University Rotterdam, and worked as
a group leader in pharmaceutical research at Organon, the Netherlands before
joining NITD in September 2003.
The Dengue Digest will continue to be published quaterly in 2006. The four
issues will centre around medicinal chemistry aspects of dengue drug discovery,
a review of advances in the molecular biology of dengue virus, comparison of
cell-based detection techniques and prospects for lab-to-lab stadardization as
well as a the status of dengue prospective studies by various groups around
the world.

Guest Editors Note:

The Dengue virus is a complex pathogen that has revealed few of its secrets.
It is still unclear what cells in the body are infected and what cells contribute
most to viral replication. It is unclear how the virus interacts with the immune
system and is able to evade it or even turn it against the person infected. But
most importantly, it is unclear why only certain people develop Dengue
Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) and how the virus
can cause these severe symptoms.
As presented in the previous issues of Dengue Digest, studies on cohorts of
patients and on in-vitro cultured human cells are underway and show great
promise in teaching us more about the disease. However, an animal model of
Dengue Fever, DHF and DSS that accurately reflects the human condition would
be of great help. Unfortunately, no animal model faithfully representing the
human disease has been identified despite many efforts. This issue of Dengue
Digest will review the animal infection systems people have developed and are
developing now to learn more about Dengue Fever and DHF in-vivo in a laboratory
controlled setting.
Dr. Johan Neyts from the Rega Institute for Medical Research in Belgium gives
an overview of the available rodent models for Dengue infection reported in the
literature. Dr. Anna Durbin from Johns Hopkins Bloomberg School of Public
Health, USA, is an expert on flavivirus infection of non-human primates and
discusses the possibilities of Dengue infection in these animals. And Dr. Ramesh
Akkina, at Colorado State University, USA, presents the potential of using
humanized animal systems for studying Dengue virus infection.
Also in this issue is a report by Dr. S. Kliks on the 2nd Asian Regional Dengue
Research Network Meeting held in Singapore end of September highlighting
many interesting new findings in Dengue research across the world.

Take a guess!

Animal models for dengue virus infections

Johan Neyts PhD Rega Institute for Medical Research, KU Leuven , Belgium.

Answers 1 (c) 2 (d) 3 (b) 4 (a)

Event Calender
Keystone Symposium: Cell Biology of Virus Entry, Replication and Pathogenesis
February 24 March 1 2006, Santa Fe, New Mexico, USA
www.keystonesymposia.org
19th International Conference on Antiviral Research
May 7-11, San Juan, Puerto Rico
www.isar-icar.com
7th Asian and Pacific Congress of Medical Virology
November 13-16, 2006. New Delhi, India
www.apcmv2006.com

Guest Editor
Dr. Wouter Schul, NITD.
Editorial Board
Ooi Eng Eong, DSO National Laboratories
Ng Lee Ching, Environmental Health Institute
Martin Hibberd, Genome Institute of Singapore
Julien Lescar, Nanyang Technological University
Mary Ng Mah Lee, National University of Singapore
Subhash Vasudevan, Novartis Institute for Tropical Diseases
Dengue Digest is a quarterly publication of the Novartis Institute for Tropical Diseases
(Co. Reg. No. : 200108104K). Please write to contact.nitd@novartis.com if you would like to receive
future editions of this newsletter.
Full details of the references used for articles in this issue are available on request. We look forward
to your valuable suggestions, contributions and feedbacks.
Graphical design by Francis Ng, Benvin Print, Singapore

Dengue can cause a variety of diseases, ranging from mild-flu like symptoms
to a disease with hemorrhagic manifestations and to a severe fatal disease
with coagulopathy and hypovolemic shock. Given the variety of symptoms and
the fact that not only viral, but also host factors (including prior infection) may
contribute to dengue pathogenesis it is understandable that it is almost impossible
to develop an animal model that would mimic all aspects of the human disease.
The development of vaccines and antiviral therapy against dengue has been
hindered by the lack of such relevant animal models.
Non-human primates are always the most relevant option for modeling a human
disease. Indeed, non-human primates show a measurable viremia after inoculation
with certain strains of Dengue virus, however they do not become ill (discussed
by Dr. Durbin in this issue.) Nonetheless, the value of the non-human primate
Dengue infection model for testing vaccine efficacy is quite well established, and
it was shown recently that anti-viral treatment can similarly be tested in the nonhuman primate system. Ajariyakhajorn et al. showed that a single injection with
pegylated recombinant IFN-2a (Pegasys, a drug used for the treatment of HCV
infections) of rhesus monkeys that had been infected with dengue serotype 2
resulted in a significant lowering of viremia and an improvement of virus clearance.

Volume 2, No. 4, December 2005

Mouse models

Although the mouse is not the natural host of the virus it may be of great
importance to have small animal models that would allow to study certain
aspects of dengue pathogenesis. Since the evaluation of antiviral agents in nonhuman primates is for obvious ethical and financial reasons limited, it will be
very important to have relevant small animals models at hand to test the efficacy
of antiviral agents. We here present a brief, non-exhaustive, overview of dengue
mouse models that have been reported in recent years.
Inbred strains

Many investigators have reported Dengue infection in specific inbred strains,

including BALB/c, C57BL/6, and A/J mice. Chen et al. could detect the Dengue
virus up to 7 days after intra-venous infection of C57BL/6 mice and observed
T-cell activation and signs of liver damage. Paes et al. found more extensive
liver injury in BALB/c mice infected with Dengue virus type 2, and Atrasheuskaya
et al. found that young BALB/c mice (of haplotype H-2d) infected with Dengue
virus type 2 develop anemia, thrombocytopenia, pre-terminal paralysis and
shock. They also showed that levels of TNF- abruptly and steeply increase
shortly before death and treatment with anti-TNF- serum can reduce mortality.
Huang et al. reported that A/J mice infected intravenously with Dengue virus
type 2 manifest thrombocytopenia and produce anti-platelet antibodies which
are suspected to contribute to some extent to Dengue Hemorrhagic Fever (DHF)
in human patients.
Genetically modified strains

Although infection of wild type inbred strains can elicit pathology the virus does
not seem to replicate to detectable levels in blood serum. Johnson and Roehrig
used mice deficient for both the interferon alpha/beta receptor and the interferon
gamma receptor, known as AG129 mice, for infection with Dengue type 2. They
found reproducible viremia in the serum and in brain and spleen. Shresta et al.
elaborated on this system to analyze the role of the interferon response in the
susceptibility of mice to Dengue infection.
SCID-hu mice

Several severe combined immuno deficient mice grafted with human cells (SCIDhu) have been infected with Dengue virus. SCID mice reconstituted with peripheral
blood lymphocytes and infected subsequently with dengue type 1 develop
viremia, although virus production was highly variable. Dengue type 2 was
shown to replicate more reliably in SCID mice transplanted with a human
hepatocarcinoma cell lines. In the early stage postinfection, virus is detected
in the liver and in serum of the engrafted mice, and at later time-points animals
develop paralysis and present with thrombocytopenia and a prolonged partial
thromboplastin time, which is a characteristic for DHF. Similarly, SCID mice that
were engrafted with a human erythroleukemia cell line (K562) and inoculated
with dengue type 2 develop paralysis and die.
Using more sophisticated SCID-hu systems, as recently reported by Bente et al.
seem to hold great promise for developing the mouse into a model for Dengue
fever and possibly DHF (discussed later in this issue by Dr. R. Akkina).

Mouse models for Dengue

Inbred laboratory strains:
Strain:
Method:
Effect:

C57BL/6, BALB/c, A/J

Intravenous injection of mouse adapted virus.
Liver damage and some thrombocytopenia, death.
No-viremia

Knockout strains:
Strain:
Method:
Effect:

AG129, STAT1-/Intravenous or intraperitoneal injection of cell culture

produced or mouse adapted virus.
Viremia, death.

Humanized SCID:
Grafted with: K562, HepG2, Huh7, CD34+ cells.
Method:
Intravenous, intraperitoneal, or subcutaneous injection
of cell culture produced virus.
Effect:
Viremia, thrombocytopenia, increased hematocrit, liver
damage, rash, death.

Dengue animal models

Humanized SCID-hu mouse models for Dengue research:
Professor Ramesh Akkina DVM,PhD - Dept. Microbiology, Immunology, and Pathology Colorado State
University, Fort Collins, Colorado, USA.

Animal models that incorporate human cells susceptible to Dengue viral infection
will have a main advantage of achieving in vivo replication of the virus in a
physiological setting. In this regard, use of immunodeficient SCID mice engrafted
with human cells or cell lines has broadened the scope of Dengue pathogenesis
studies. Infection of SCID mice engrafted with human hepatocarcinoma (HepG2)
or erythroleukemia cells (K562) lead to sustained high viral load and recapitulated
some aspects of dengue pathogenesis. In a recent development, immunodeficient
NOD-SCID mice engrafted with human cord blood hematopoietic progenitor
cells gave rise to development of a full repertoire of human target dendritic
cells in vivo. When these mice were infected with Dengue virus, clinical signs
typical of Dengue fever namely, fever, rash and thrombocytopenia were seen.
There was marked in vivo replication of the virus coinciding with clinical signs.
Although encouraging and useful for potential therapeutic drug testing, these
models fall short for vaccine evaluations and safety testing to prevent antibody
dependant enhancement of infection. As these models lack a full complement
of circulating immunocompetent cells, they are unable to coordinate a human
immune response. However, recent technical innovations in the creation of
improved SCID-hu mouse models capable of primary and secondary immune
responses have paved the way for exciting new avenues of investigation.
In SCID-hu immunocompetent (IC) model, mice are transplanted with human
fetal bone marrow, thymus, skin fragments, and mesenteric lymph nodes. T and

B cells develop from the bone marrow and thymus. The skin grafts become
vascularized and seeded with human cells. When these IC mice are immunized
with tetanus toxoid, both antigen specific T and B cell responses are vigorously
elicited. As all the cell types needed for proper immune response including T
cells, B cells, monocytes/macrophages, dendritic cells are integral to this model,
it has great potential for modeling Dengue viral replication and pathogenesis.
Combination of engrafted human lymphatic organs and skin should provide the
microenvironment to initiate viral replication at the skin entry site followed by
viral spread to other cells. Infected mice could potentially develop a balanced
infection-immune response essential for investigating dengue pathogenesis and
immune enhancement. A particular advantage is that dendritic and monocytic
cells are continuously produced, thus providing fresh target cells for the virus.
In addition to this model, a new development is the Rag2 common gamma chain
double knock out mouse model transplanted with human cord blood derived
CD34 hematopoietic progenitor cells. Following engraftment, mice develop good
levels of human B cells, T cells, monocytes, and dendritic cells. Organized
structures of primary and secondary lymphoid organs are seen. Most importantly,
human immune responses are generated when these mice are vaccinated with
tetanus toxoid and infected with Epstein-Barr virus. Given the presence of
relevant human cells such as monocytes and dendritic cells in the system and
its capacity to give an immune response, this model also shows great promise
for Dengue virus work. These promising humanized IC mouse models are currently
being investigated our laboratory of at Colorado State University in Fort Collins.

CD4+
CD8+
Treg (Foxp3+)

Graft fetal
thymus, bone
marrow, skin,
lymph nodes

B cell compartment

Granulocytes

IgM
IgG

Total and
antigen
specific

Lymphocytes
Dendritic cells
Plasmacytoid
RAG2-/- c-/- or SCID

A major obstacle to understanding the etiology of dengue-induced illness is the

absence of an animal model that reliably reproduces human disease. In nature,
Dengue viruses are limited to three natural hosts: mosquitoes, various nonhuman primates, and humans. Non-human primates are thought to be the primary
mammalian hosts of Dengue virus during sylvatic transmission in rural areas.
Because of the ability of Dengue virus to replicate in non-human primates,
several non-human primate species have been experimentally infected with the
virus and evaluated as animal models for Dengue-induced disease.
Chimpanzees

Chimpanzees infected with low-passaged Dengue virus isolates obtained from

patients with Dengue Hemorrhagic Fever (DHF) or Dengue Shock Syndrome
(DSS) developed viremias lasting from five to nine days. The mean peak titers
of Dengue virus in the chimpanzees were lower than that typically seen in
patients with DHF/DSS, and none of the animals developed clinical or laboratory
evidence of Dengue fever or DHF. Humans who get re-infected with a Dengue
serotype different from the first infection are more likely to develop severe
DHF/DSS through a mechanism called antibody dependent enhancement (ADE).
To investigate whether a similar phenomenon could be observed in the chimpanzee
model a subset of chimpanzees was challenged with heterotypic virus 26 months
after primary infection. Three out of four animals became viremic, but no animal
developed overt disease.

When it comes to Dengue pathology the macaque showed more significant

differences to the human disease. Modest axillary and inguinal lymphadenopathy
was noted four to ten days after primary Dengue infection in macaques, but no
overt illness was ever observed. Of the more than 100 macaques challenged
with a heterologous Dengue virus to elicit an ADE effect, no animal developed
a classic DHF/DSS syndrome. However, there was a significant increase in peak
viremia titers during secondary DEN-2 infection compared to primary infection.
Additionally, one animal that received DEN-2 challenge three months after a
primary DEN-4 infection developed some laboratory abnormalities associated
with DHF including thrombocytopenia, elevated prothrombin time, decrease in
complement level, and an early unsustained elevation in hematocrit.
Although the macaque model has yet to be optimized as a model for DHF/DSS,
it has proven to be extraordinarily useful for vaccine development. Because
macaques develop detectable levels of viremia when infected with DENV,
attenuation of live vaccine candidate viruses can be assessed in the macaque
model. Reduction in peak virus titer in the rhesus macaque is an important
marker of attenuation for a live candidate Dengue vaccine.

Owl monkeys (Aotus nancymae) can be infected with

Dengue and occasionally show mild clinical symptoms

Owl monkeys were able to be infected experimentally with Dengue virus, but
their susceptibility to the different Dengue serotypes was not consistent.
Interestingly, six of the twenty infected Owl monkeys did develop neutropenia.
In addition, some clinical symptoms (lethargy and decreased appetite) were
described in two of the infected Owl monkeys but one of these was never viremic.
Macaques

Rhesus macaques (Macaca mulatta) have been the most extensively utilized
non-human primate model for Dengue. Halstead et al. infected hundreds of
macaques to determine patterns of viremia, antibody response, and the
pathogenesis of the Dengue virus. These studies determined that macaques
can be infected with all four serotypes of Dengue virus at doses ranging from
quite low (< 10 PFU) to as high as 50,000 PFU. Additionally, they showed that
monocytes appeared to be the cells in the peripheral blood that are permissive
for Dengue virus infection. The onset of viremia, duration of viremia, and
serological response to Dengue virus in macaques is very similar to that observed
in humans, although the peak in virus titers were generally lower in macaques
than in humans.

Myeloid
Dendritic cells
Myeloid

Transfer CD34+ stem

cells from cord blood

Anna Durbin PhD - Johns Hopkins Bloomberg School of Public Health, Baltimore, USA.

Owl Monkeys

T cell compartment
Sublethal
irradiation

Dengue Infection in non-human primates

Grafting severe immunocompromised mice like SCID or

RAG2-/-c-/- with human fetal lymphatic tissue or
hematopoietic stem cells results in animals with a human
immune system, including a full T-cell repertoire, B-cells
capable of isotype class switching, and dendritic cells.

Phagocytes
Mononuclear

New Treatment Strategies for Dengue and other Flaviviral Diseases

NOVARTIS FOUNDATION SYMPOSIUM 277
This book proceeds from a meeting which brought together the
worlds leading dengue and flaviviral disease researchers, in order
to examine the current state of the art in the molecular biology of
the dengue virus. There is a particular emphasis on structure and

function of the virus and on the targeting of virus proteins by potential

anti-viral agents. Data on the pathogenesis of dengue and dengue
haemorrhagic fever are discussed in detail, including target cells and
receptors, in order to develop a clear view of the host and viral factors

ORDERING INFORMATION
Estimated publication date: March 2006
ISBN: 0470016434
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NOVARTIS FOUNDATION SYMPOSIA

This volume contains the proceedings of a Novartis Foundation symposium.
The extensive and stimulating discussions following the presentation of
papers form an integral part of the book and have earned these volumes
a unique and respected place in scientific literature.
www.novartisfound.org.uk

that contribute to dengue haemorrhagic fever. Finally, the book

reviews the options for treatment of dengue and dengue haemorrhagic
fever, paying particular attention to ways in which vector, host and
environment can play a critical role in the spread of this disease.

The Novartis Foundation

promoting excellence in science worldwide

Fighting flaviviruses
By Lisa Melton, Science writer at the Novartis Foundation, London.

Novartis Foundation Symposium 277 was held in collaboration with the Novartis
Institute for Tropical Diseases in Singapore(26-27 September, 2005)
It was a timely occasion. As the worlds leading dengue researchers gathered
last September for a symposium on new treatment strategies for dengue and
other flaviviral diseases, Singapore was fighting the worst dengue outbreak in
its history. The need is urgent. The whole of the tropical world is involved. Few
places are free from Dengue, said Duane Gubler, from the University of Hawaii,
USA, kick-starting the talks.
Dengue has plagued humans since antiquity yet it has languished in one of
sciences backwaters. Now Dengue fever is firmly back on the research radar,
and the challenge is to decide how best to tackle this unique pathogen. The
symposium, organised by the London-based Novartis Foundation in collaboration
with Singapores NITD, brought together molecular biologists, clinicians, medicinal
chemists and epidemiologists to map the best strategies leading to new, muchneeded anti-virals and vaccines.
Drug discovery for any disease is a long and arduous process. And as Thomas
Keller, a medicinal chemist at NITD, reminded symposiasts Dengue fever has
the additional complication that its target population is young and lives in
developing countries.
The ideal drug should hit a viral protein that is essential for its survival. Julien
Lescar from the Singapore School of Biological Sciences, in collaboration with

the NITD team, has scrutinized the NS3 protein using site-directed mutagenesis.
Acting on Lescars finding, NITD scientists are wading through libraries of small
molecules searching for compounds that clamp down on the NS3 helicase.
The enigma of why only some dengue-infected people develop life-threatening
symptoms remains unresolved. According to Jeremy Farrar, a clinical researcher
at the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam what is
needed is a fish and chips study to pinpoint whether it is virus virulence or
host genetic susceptibility or both.
Some answers may well arise from the EDEN (Early Dengue) study. Martin
Hibberd, at the Genome Institute of Singapore has begun tracking hundreds of
patients attending the citys largest hospital. The scientists will sequence the
entire viral genome in each infection, using a chip-based approach. In parallel,
they will follow 24,000 genes in each patients DNA as they respond to dengue
infection. Variations in peoples responses can then be linked to genetic differences.
Gavin Screaton, a clinician and researcher at the Hammersmith Hospital, London,
UK pointed out Dengue is an exceptional pathogen because the disease is
usually worse the second time round. This is a critical issue for vaccine developers
to resolve, since an immune-boosting therapy could trigger severe disease when
the person becomes infected.
A silver-bullet is unlikely, participants agreed. But a multi-pronged attack on
the virus, that combines anti-virals and vaccines, has a good chance of success.